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BACKGROUND: We investigated the prognostic role of tumour-associated macrophages (TAMs) in patients with head and neck squamous cell carcinoma (HNSCC) treated with definitive chemoradiotherapy (CRT). METHODS: The expression of CD68+, CD163+ and CD11b+ cells was assessed using immunohistochemistry in n=106 pre-treatment tumour biopsy samples and was correlated with clinicopathological characteristics, including T-stage, N-stage, grading, tumour localisation, age and sex as well as local failure-free survival (LFFS), distant metastases-free survival (DMFS), progression-free (PFS), and overall survival (OS). Finally, TAMs expression and vessel density (CD31) were examined in n=12 available early local recurrence samples and compared with their matched primary tumours . The diagnostic images and radiotherapy plans of these 12 patients were also analysed. All local recurrences occurred in the high radiation dose region (⩾70 Gy). RESULTS: With a median follow-up of 40 months, OS at 2 years was 60.5%. High CD163 expression in primary tumours was associated with decreased OS (P=0.010), PFS (P=0.033), LFFS (P=0.036) and DMFS (P=0.038) in multivariate analysis. CD163 demonstrated a strong prognostic value only in human papillomavirus (p16(INK4))-negative patients. Early local recurrence specimens demonstrated a significantly increased infiltration of CD11b+ myeloid cells (P=0.0097) but decreased CD31-positive vessel density (P=0.0004) compared with their matched primary samples. CONCLUSIONS: Altogether, baseline CD163 expression predicts for an unfavourable clinical outcome in HNSCC after definitive CRT. Early local recurrences showed increased infiltration by CD11b+ cells. These data provide important insight on the role of TAMs in mediating response to CRT in patients with HNSCC.

New oral anticoagulants for the prevention of stroke and systemic embolism in patients with atrial fibrillation have recently been introduced. In this translational study, we explored the risk of long-term anticoagulation on intracerebral hemorrhage under sustained severe arterial hypertension. We initiated anticoagulation with warfarin or apixaban in spontaneously hypertensive rats prone to develop severe hypertension and subsequent intracerebral bleeding complications. A non-anticoagulated group served as control. During an 11-week-study period, blood pressure, anticoagulation parameters, and clinical status were determined regularly. The incidence of histopathologically proven intracerebral hemorrhage was defined as the primary endpoint. Both warfarin and apixaban anticoagulation was fairly stable during the study period, and all rats developed severe hypertension. Intracerebral hemorrhage was determined in 29% (4/14) of warfarin rats and in 10% (1/10) of apixaban rats. Controls did not show cerebral bleeding complications (chi-square not significant). Mortality rate at study termination was 33% (2/6) in controls, 43% (6/14) in the warfarin group, and 60% (6/10) in the apixaban group. Animals died from extracerebral complications in most cases. Our study describes an experimental intracerebral hemorrhage model in the context of sustained hypertension and long-term anticoagulation. Extracerebral bleeding complications occurred more often in warfarin-treated animals compared with apixaban and control rats.

Mutations of the human plectin gene (PLEC) on chromosome 8q24 cause autosomal recessive epidermolysis bullosa simplex with muscular dystrophy (EBS-MD). In the present study we analyzed the downstream effects of PLEC mutations on plectin protein expression and localization, the structure of the extrasarcomeric desmin cytoskeleton, protein aggregate formation and mitochondrial distribution in skeletal muscle tissue from three EBS-MD patients. PLEC gene analysis in a not previously reported 35-year-old EBS-MD patient with additional disease features of cardiomyopathy and malignant arrhythmias revealed novel compound heterozygous (p.(Phe755del) and p.(Lys1040Argfs*139)) mutations resulting in complete abolition of plectin protein expression. In contrast, the other two patients with different homozygous PLEC mutations showed preserved plectin protein expression with one only expressing rodless plectin variants, and the other markedly reduced protein levels. Analysis of skeletal muscle tissue from all three patients revealed severe disruption of the extrasarcomeric intermediate filament cytoskeleton, protein aggregates positive for desmin, syncoilin, and synemin, degenerative myofibrillar changes, and mitochondrial abnormalities comprising respiratory chain dysfunction and an altered organelle distribution and amount. Our study demonstrates that EBS-MD causing PLEC mutations universally result in a desmin protein aggregate myopathy phenotype despite marked differences in individual plectin protein expression patterns. Since plectin is the key cytolinker protein that regulates the structural and functional organization of desmin filaments, the defective anchorage and spacing of assembled desmin filaments is the key pathogenetic event that triggers the formation of desmin protein aggregates as well as secondary mitochondrial pathology. Electronic supplementary material The online version of this article (doi:10.1186/s40478-016-0314-7) contains supplementary material, which is available to authorized users.

Background Ovarian cancer (OvCA) tissues show abundant expression of the ectonucleotidases CD39 and CD73 which generate immunomodulatory adenosine, thereby inhibiting cytotoxic lymphocytes. Little, however, is known about the effect of adenosine on myeloid cells. Considering that tumor associated macrophages (TAM) and myeloid-derived suppressor cells (MDSC) constitute up to 20 % of OvCA tissue, we investigated the effect of adenosine on myeloid cells and explored a possible contribution of myeloid cells to adenosine generation in vitro and ex vivo. Methods Monocytes were used as human blood-derived myeloid cells. After co-incubation with SK-OV-3 or OAW-42 OvCA cells, monocyte migration was determined in transwell assays. For conversion into M2-polarized “TAM-like” macrophages, monocytes were co-incubated with OAW-42 cells. Ex vivo TAMs were obtained from OvCA ascites. Macrophage phenotypes were investigated by intracellular staining for IL-10 and IL-12. CD39 and CD73 expression were assessed by FACS analysis both on in vitro-induced TAM-like macrophages and on ascites-derived ex situ-TAMs. Myeloid cells in solid tumor tissue were analyzed by immunohistochemistry. Generation of biologically active adenosine by TAM-like macrophages was measured in luciferase-based reporter assays. Functional effects of adenosine were investigated in proliferation-experiments with CD4+ T cells and specific inhibitors. Results When CD39 or CD73 activity on OvCA cells were blocked, the migration of monocytes towards OvCA cells was significantly decreased. In vivo, myeloid cells in solid ovarian cancer tissue were found to express CD39 whereas CD73 was mainly detected on stromal fibroblasts. Ex situ-TAMs and in vitro differentiated TAM-like cells, however, upregulated the expression of CD39 and CD73 compared to monocytes or M1 macrophages. Expression of ectonucleotidases also translated into increased levels of biologically active adenosine. Accordingly, co-incubation with these TAMs suppressed CD4+ T cell proliferation which could be rescued via blockade of CD39 or CD73. Conclusion Adenosine generated by OvCA cells likely contributes to the recruitment of TAMs which further amplify adenosine-dependent immunosuppression via additional ectonucleotidase activity. In solid ovarian cancer tissue, TAMs express CD39 while CD73 is found on stromal fibroblasts. Accordingly, small molecule inhibitors of CD39 or CD73 could improve immune responses in ovarian cancer. Electronic supplementary material The online version of this article (doi:10.1186/s40425-016-0154-9) contains supplementary material, which is available to authorized users.

Biomarkers are strongly needed for diagnostic surveillance of patients with metastatic melanoma. On the basis of its known association with tumor metastasis and its ability to induce cancer cachexia, we investigated serum levels of growth and differentiation factor 15 (sGDF-15) as a marker for overall survival (OS). sGDF-15 was retrospectively measured by ELISA in 761 samples obtained at distinct time points during routine clinical care of patients with stage III/IV melanoma. In the entire cohort, sGDF-15 ≥ 1.5 ng/ml was strongly associated with reduced OS after assessment. Subsequent analyses were performed separately for tumor-free stage III, tumor-free stage IV, and unresectable stage IV patients. For patients with unresectable distant metastasis (n = 206), sGDF-15 was independently associated with OS when considered together with the M-category and superior to serum level of lactate dehydrogenase. Analysis in tumor-free stage III patients during routine surveillance (n = 468) revealed sGDF-15 to be associated with OS and an independent factor when considered together with S100B and the pattern of locoregional metastasis. Only in tumor-free stage IV patients (n = 87) sGDF-15 was not associated with OS. sGDF-15 should thus be further validated as a marker for early detection of recurrence in stage III patients and as a prognostic or predictive marker particularly in the context newly available treatments in unresectable stage IV patients.

Leptomeningeal dissemination of a primary brain tumor is a condition which is challenging to treat, as it often occurs in rather late disease stages in highly pretreated patients. Its prognosis is dismal and there is still no accepted standard of care. We report here a good clinical effect with a partial response in three out of nine patients and a stable disease with improvement on symptoms in two more patients following systemic anti-angiogenic treatment with bevacizumab (BEV) alone or in combination with chemo- and/or radiotherapy in a series of patients with leptomeningeal dissemination from primary brain tumors (diffuse astrocytoma WHO°II, anaplastic astrocytoma WHO°III, anaplastic oligodendroglioma WHO°III, primitive neuroectodermal tumor and glioblastoma, both WHO°IV). This translated into effective symptom control in five out of nine patients, but only moderate progression-free and overall survival times were reached. Partial responses as assessed by RANO criteria were observed in three patients (each one with anaplastic oligodendroglioma, primitive neuroectodermal tumor and glioblastoma). In these patients progression-free survival (PFS) intervals of 17, 10 and 20 weeks were achieved. In three patients (each one with diffuse astrocytoma, anaplastic astrocytoma and primitive neuroectodermal tumor) stable disease was observed with PFS of 13, 30 and 8 weeks. Another three patients (all with glioblastoma) were primary non-responders and deteriorated rapidly with PFS of 3 to 4 weeks. No severe adverse events were seen. These experiences suggest that the combination of BEV with more conventional therapy schemes with chemo- and/or radiotherapy may be a palliative treatment option for patients with leptomeningeal dissemination of brain tumors.

Objective— Reactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidases contribute to angiogenesis and vascular repair. NADPH oxidase organizer 1 (NoxO1) is a cytosolic protein facilitating assembly of constitutively active NADPH oxidases. We speculate that NoxO1 also contributes to basal reactive oxygen species formation in the vascular system and thus modulates angiogenesis. Approach and Results— A NoxO1 knockout mouse was generated, and angiogenesis was studied in cultured cells and in vivo. Angiogenesis of the developing retina and after femoral artery ligation was increased in NoxO1 −/− when compared with wild-type animals. Spheroid outgrowth assays revealed greater angiogenic capacity of NoxO1 −/− lung endothelial cells (LECs) and a more tip-cell–like phenotype than wild-type LECs. Usually signaling by the Notch pathway switches endothelial cells from a tip into a stalk cell phenotype. NoxO1 −/− LECs exhibited attenuated Notch signaling as a consequence of an attenuated release of the Notch intracellular domain on ligand stimulation. This release is mediated by proteolytic cleavage involving the α-secretase ADAM17. For maximal activity, ADAM17 has to be oxidized, and overexpression of NoxO1 promoted this mode of activation. Moreover, the activity of ADAM17 was reduced in NoxO1 −/− LECs when compared with wild-type LECs. Conclusions— NoxO1 stimulates α-secretase activity probably through reactive oxygen species–mediated oxidation. Deletion of NoxO1 attenuates Notch signaling and thereby promotes a tip-cell phenotype that results in increased angiogenesis.

BACKGROUND Choroid plexus tumors are intraventricular neoplasms derived from the choroid plexus epithelium. A better knowledge of molecular factors involved in choroid plexus tumor biology may aid in identifying patients at risk for recurrence. METHODS Methylation profiles were examined in 29 choroid plexus papillomas (CPPs, WHO grade I), 32 atypical choroid plexus papillomas (aCPPs, WHO grade II), and 31 choroid plexus carcinomas (CPCs, WHO grade III) by Illumina Infinium HumanMethylation450 Bead Chip Array. RESULTS Unsupervised hierarchical clustering identified 3 subgroups: methylation cluster 1 (pediatric CPP and aCPP of mainly supratentorial location), methylation cluster 2 (adult CPP and aCPP of mainly infratentorial location), and methylation cluster 3 (pediatric CPP, aCPP, and CPC of supratentorial location). In methylation cluster 3, progression-free survival (PFS) accounted for a mean of 72 months (CI, 55-89 mo), whereas only 1 of 42 tumors of methylation clusters 1 and 2 progressed (P< .001). On stratification of outcome data according to WHO grade, all CPCs clustered within cluster 3 and were associated with shorter overall survival (mean, 105 mo [CI, 81-128 mo]) and PFS (mean, 55 mo [CI, 36-73 mo]). The aCPP of methylation cluster 3 also progressed frequently (mean, 69 mo [CI, 44-93 mo]), whereas no tumor progression was observed in aCPP of methylation clusters 1 and 2 (P< .05). Only 1 of 29 CPPs recurred. CONCLUSIONS Methylation profiling of choroid plexus tumors reveals 3 distinct subgroups (ie, pediatric low-risk choroid plexus tumors [cluster 1], adult low-risk choroid plexus tumors [cluster 2], and pediatric high-risk choroid plexus tumors [cluster 3]) and may provide useful prognostic information in addition to histopathology.

TALE-homeodomain proteins function as part of heteromeric complexes that contain one member each of the PBC- and MEIS/PREP-subclasses. As we have recently shown, MEIS2 cooperates with the neurogenic transcription factor PAX6 in the control of adult subventricular zone (SVZ) neurogenesis in rodents. Expression of the PBC-protein Pbx1 in the SVZ has been reported but its functional role(s) had not yet been investigated. Using a genetic loss-of-function model, we now show that Pbx1 is an early regulator of SVZ neurogenesis. Targeted deletion of Pbx1 by retroviral transduction of Cre recombinase into Pbx2-deficient SVZ stem- and progenitor cells carrying floxed alleles of Pbx1 significantly reduced the production of neurons and increased the generation of oligodendrocytes. Loss of Pbx1-expression in neuronally committed neuroblasts in the rostral migratory stream in a Pbx2 null (Pbx2−/−) background, by contrast, severely compromised cell survival. By chromatin immunoprecipitation from endogenous tissues or isolated cells, we further detect PBX1 binding to known regulatory regions of the neuron-specific genes DCX and TH days or even weeks before the respective genes are expressed during the normal program of SVZ neurogenesis, suggesting that PBX1 may act as priming factor to mark these genes for subsequent activation. Collectively, our results establish that PBX1 regulates adult neural cell fate determination in a way that goes beyond the that of its heterodimerization partner MEIS2.

Diffuse growth of gliomas is based on enhanced cell migration and remodeling of the extracellular matrix. Up-regulation of matrix metalloproteinases in gliomas is associated with a poor prognosis. The activated leukocyte adhesion molecule is considered to be indispensable for conversion of matrix metalloproteinase 2 into its active form. We therefore investigated the expression of activated leukocyte adhesion molecule in 9 malignant glial cell lines, 105 normal/reactive human brain specimens, 248 astrocytomas/glioblastomas, 98 ependymomas, 35 oligodendrogliomas, 10 neurocytomas, 10 primitive neuroectodermal tumors (PNET), and 36 medulloblastomas by immunohistochemistry and in selected cases by reverse transcriptase polymerase chain reaction. Correlation between activated leukocyte adhesion molecule expression and tumor grades and entities, proliferation activity, matrix metalloproteinase 2 expression, prognostic isocitrate dehydrogenase (IDH)1 mutation (R132H) status, O-6-methylguanine DNA-methyltransferase (MGMT) promoter status, or association with patient survival were analyzed. All oligodendrogliomas were strongly activated leukocyte adhesion molecule positive. Numbers of activated leukocyte adhesion molecule positive tumors were higher in glioblastomas (93%) than in diffuse astrocytomas (83%), but mean expression intensity was significantly reduced. Anaplastic ependymomas (68%) exhibited reduced numbers of activated leukocyte adhesion molecule-positive tumors and staining intensity compared with lower-grade ependymomas (85%). Activated leukocyte adhesion molecule expression in gliomas was independent of proliferative activity, MGMT status, patient survival, and age, whereas gliomas with IDH1 (R132H) mutation had significantly higher activated leukocyte adhesion molecule levels than their wild-type counterparts. Matrix metalloproteinase 2-negative glioblastomas exhibited significantly reduced activated leukocyte adhesion molecule expression levels compared with astrocytomas. In summary, our findings indicate that activated leukocyte adhesion molecule expression levels in gliomas are probably linked to other mechanisms than its supposed role as regulator of matrix metalloproteinase 2.

Glioblastoma may develop rapidly without evidence for precursor lesions (primary glioblastomas), or progress from diffuse or anaplastic astrocytomas (secondary glioblastomas). Despite having distinct genetic profiles, these glioblastoma subtypes have similar histological features. We hypothesized that the highly malignant phenotype of glioblastoma may be attributable to genetic alterations that are common to both glioblastoma subtypes. In the present study, we first searched for commonly (>35%) amplified genes in glioblastomas with IDH1 mutation (a hallmark of secondary glioblastoma) and those without IDH1 mutation (typical for primary glioblastoma) in data from The Cancer Genome Atlas (TCGA). A total of 25 genes were identified, of which 21 were located at 7q31-34. We then screened 264 gliomas (70 glioblastomas, 112 diffuse astrocytomas, 82 oligodendrogliomas) for gain of the MET at 7q31.2 with quantitative polymerase chain reaction (PCR). MET gain was detected in primary glioblastomas (47%) and secondary glioblastomas (44%), suggesting that this genetic alteration plays a role in the pathogenesis of both glioblastoma subtypes. MET gain was also common in diffuse astrocytomas (38%), but less frequent in oligodendrogliomas (16%). MET gain in diffuse astrocytomas was associated with shorter survival (median, 43.0 vs. 70.7 months; P = 0.004), suggesting that MET gain is a useful prognostic marker for diffuse astrocytomas.

Background Cardiac ischemia-reperfusion (I-R) injury represents a major cause of cardiac tissue injury. Adenosine signaling dampens inflammation during cardiac I-R. The authors investigated the role of the adenosine A2b-receptor (Adora2b) on inflammatory cells during cardiac I-R. Methods To study Adora2b signaling on inflammatory cells, the authors transplanted wild-type (WT) bone marrow (BM) into Adora2b(-/-) mice or Adora2b(-/-) BM into WT mice. To study the role of polymorphonuclear leukocytes (PMNs), neutrophil-depleted WT mice were treated with an Adora2b agonist. After treatments, mice were exposed to 60 min of myocardial ischemia and 120 min of reperfusion. Infarct sizes and troponin I concentrations were determined by triphenyltetrazolium chloride staining and enzyme-linked immunosorbent assay, respectively. Results Transplantation of WT BM into Adora2b(-/-) mice decreased infarct sizes by 19 ± 4% and troponin I by 87.5 ± 25.3 ng/ml (mean ± SD, n = 6). Transplantation of Adora2b(-/-) BM into WT mice increased infarct sizes by 20 ± 3% and troponin I concentrations by 69.7 ± 17.9 ng/ml (mean ± SD, n = 6). Studies on the reperfused myocardium revealed PMNs as the dominant cell type. PMN depletion or Adora2b agonist treatment reduced infarct sizes by 30 ± 11% or 26 ± 13% (mean ± SD, n = 4); however, the combination of both did not produce additional cardioprotection. Cytokine profiling showed significantly higher cardiac tumor necrosis factor α concentrations in Adora2b(-/-) compared with WT mice (39.3 ± 5.3 vs. 7.5 ± 1.0 pg/mg protein, mean ± SD, n = 4). Pharmacologic studies on human-activated PMNs revealed an Adora2b-dependent tumor necrosis factor α release. Conclusion Adora2b signaling on BM-derived cells such as PMNs represents an endogenous cardioprotective mechanism during cardiac I-R. The authors' findings suggest that Adora2b agonist treatment during cardiac I-R reduces tumor necrosis factor α release of PMNs, thereby dampening tissue injury.

Chromosomal 7q34 duplication and BRAF-KIAA1549 fusion is a characteristic genetic alteration in pilocytic astrocytomas. 7q34 gain appears to be common in diffuse astrocytomas, but its significance is unclear. We assessed BRAF gain and BRAF mutations in 123 low-grade diffuse gliomas, including 55 diffuse astrocytomas, 18 oligoastrocytomas and 50 oligodendrogliomas. Quantitative polymerase chain reaction (PCR) revealed BRAF gain in 17/50 (34%) oligodendrogliomas, a significantly higher frequency than in diffuse astrocytomas (7/55; 13%; P = 0.0112). BRAF gain was common in low-grade diffuse gliomas with 1p/19q loss (39%) and those lacking any of the genetic alterations analyzed (31%), but was rare in those with TP53 mutations (2%). Logistic regression analysis showed a significant positive association between 1p/19q loss and BRAF gain (P = 0.0032) and a significant negative association between TP53 mutations and BRAF gain (P = 0.0042). Fluorescence in situ hybridization (FISH) analysis of 26 low-grade diffuse gliomas with BRAF gain additionally revealed BRAF-KIAA1549 fusion in one oligodendroglioma. Sequencing of cDNA in 17 low-grade diffuse gliomas showed BRAF-KIAA1549 fusion in another oligodendroglioma. A BRAF(V600E) mutation was also detected in one oligodendroglioma, and a BRAF(A598V) in one diffuse astrocytoma. These results suggest that low-grade diffuse gliomas with 1p/19q loss have frequent BRAF gains, and a small fraction of oligodendrogliomas may show BRAF-KIAA1549 fusion.

Medulloblastomas account for 20% of pediatric brain tumors. With an overall survival of 40%–70%, their treatment is still a challenge. The majority of medulloblastomas lack p53 mutations, but even in cancers retaining wild-type p53, the tumor surveillance function of p53 is inhibited by the oncoprotein MDM2. Deregulation of the MDM2/p53 balance leads to malignant transformation. Here, we analyzed MDM2 mRNA and protein expression in primary medulloblastomas and normal cerebellum and assessed the mutational status of p53 and MDM2 expression in 6 medulloblastoma cell lines. MDM2 expression was elevated in medulloblastomas, compared with cerebellum. Four of 6 medulloblastoma cell lines expressed wild-type p53 and high levels of MDM2. The tumor-promoting p53-MDM2 interaction can be inhibited by the small molecule, nutlin-3, restoring p53 function. Targeting the p53-MDM2 axis using nutlin-3 significantly reduced cell viability and induced either cell cycle arrest or apoptosis and expression of the p53 target gene p21 in these 4 cell lines. In contrast, DAOY and UW-228 cells harboring TP53 mutations were almost unaffected by nutlin-3 treatment. MDM2 knockdown in medulloblastoma cells by siRNA mimicked nutlin-3 treatment, whereas expression of dominant negative p53 abrogated nutlin-3 effects. Oral nutlin-3 treatment of mice with established medulloblastoma xenografts inhibited tumor growth and significantly increased survival. Thus, nutlin-3 reduced medulloblastoma cell viability in vitro and in vivo by re-activating p53 function. We suggest that inhibition of the MDM2-p53 interaction with nutlin-3 is a promising therapeutic option for medulloblastomas with functional p53 that should be further evaluated in clinical trials.

Several methods have been introduced to improve the extent of resection in glioma surgery. Yet, radical tumor resections must not be attempted at the cost of neurological deterioration. We sought to assess whether the use of an intraoperative MRI (iMRI) in combination with multimodal neurophysiological monitoring is suitable to increase the extent of resection without endangering neurological function in patients with eloquently located gliomas. Fifty-four patients were included in this study. In 21 patients (38.9 %), iMRI led to additional tumor resection. A radiologically complete resection was achieved in 31 patients (57.4 %), while in 12 of these, iMRI had depicted residual tumor tissue before resection was continued. The mean extent of resection was 92.1 % according to volumetric analyses. Postoperatively, 13 patients (24.1 %) showed new or worsening of pre-existing sensory motor deficits. They were severe in 4 patients (7.4 %). There was no correlation between the occurrence of either any new (P = 0.77) or severe (P = 1.0) sensory motor deficit and continued resection after intraoperative image acquisition. Likewise, tumor location, histology, and tumor recurrence did not influence complication rate on uni- and multivariate analysis. We conclude that the combination of iMRI guidance with multimodal neurophysiological monitoring allows for extended resections in glioma surgery without inducing higher rates of neurological deficits, even in patients with eloquently located tumors.

Glioblastoma (GBM), the most common malignant brain tumor, is among the most lethal neoplasms, with a median survival of approximately 1 year. Prognosis is poor since GBMs possess a strong migratory and highly invasive potential, making complete surgical resection impossible. Reduced expression of carboxypeptidase E (CPE), a neuropeptide-processing enzyme, in a cell death-resistant glioma cell line and lower CPE expression levels in the cohort of GBM samples of The Cancer Genome Atlas compared to normal brain control specimens prompted us to analyze the function of CPE as a putative tumor suppressor gene. In our samples, CPE was also reduced in GBM compared to normal brain with the strongest loss in cells surrounding hypoxic tumor areas as well as in most glioma cell lines and primary glioma cells. In our cohort of glioma patients, loss of CPE predominantly occurred in glioblastomas and was associated with worse prognosis. In glioma cells, CPE overexpression was significantly reduced, whereas knockdown or inhibition enhanced glioma cell migration and invasion. The decreased migratory potential following CPE overexpression was paralleled by altered cellular morphology, promoting a transition to focal adhesions and associated stress fibers. In contrast to the decreased migration, high CPE levels were associated with higher proliferative rates. As microenvironmental regulation cues, we identified CPE as being downregulated upon hypoxia or glucose deprivation. Our findings indicate an oxygen- and nutrition-dependent anti-migratory, but pro-proliferative role of CPE in gliomas with prognostic impact for patient survival, thereby contributing to the understanding of the "go or grow" hypothesis in gliomas.

AVEN has been identified as an inhibitor of apoptosis, which binds to the adaptor protein, APAF-1, and thereby prevents apoptosome formation and mitochondrial apoptosis. Recent data have demonstrated high expression levels of AVEN messenger RNA in acute leukemias as well as a positive correlation between AVEN mRNA overexpression and poor prognosis in childhood acute lymphoblastic leukemia. On the basis of these data, we investigated the potential involvement of AVEN in tumorigenesis. First, we confirmed the overexpression of AVEN in T-cell acute lymphoblastic leukemia/lymphoma (T-ALL) patient samples. We then established a transgenic mouse model with T-cell-specific overexpression of AVEN, with which we demonstrated the oncogenic cooperation of AVEN with heterozygous loss of p53. Finally, we used a subcutaneous xenograft mouse model to show that AVEN knockdown in the T-ALL cell lines, MOLT-4 and CCRF-CEM, and in the acute myeloblastic leukemia cell line, Kasumi-1, leads to a halt in tumor growth owing to the increased apoptosis and decreased proliferation of tumor cells. Collectively, our data demonstrate that the anti-apoptotic molecule, AVEN, functions as an oncoprotein in hematopoietic neoplasms.

Anti-angiogenetic therapy is widely used in patients with glioma in the relapse situation. In glioblastoma and glioma, bevacizumab has been shown to have an impact on progression-free survival and neurologic function, but not on overall survival. However, its use on patients with gliomas showing a gliomatosis cerebri growth pattern is still more controversial. Due to the mainly diffuse infiltrative growth of these tumors, it may appear questionable whether bevacizumab can have a therapeutic effect in patients with these tumors. However, the development of nodular, necrotic and/or contrast-enhancing lesions in patients with a gliomatosis cerebri pattern is not uncommon and may indicate active neo-angiogenesis. Therefore, control of growth of these lesions as well as control of edema and reduction of steroid use may be viewed as rationales for the use of bevacizumab in these patients. In this patient series, we report on seventeen patients with primary brain tumors and a gliomatosis cerebri growth pattern (seven glioblastoma WHO°IV, two anaplastic astrocytoma WHO°III, one anaplastic oligodendroglioma WHO°III, seven astrocytoma WHO°II) treated with bevacizumab alone or in combination with lomustine or irinotecan. We found bevacizumab to be active with response rates, progression-free survival and overall survival intervals comparable to a control cohort of patients with gliomas of a less infiltrative phenotype. Based on these results, anti-angiogenic therapy with bevacizumab should also be considered in patients suffering from gliomas of a mainly infiltrative phenotype.

In human inflammatory diseases, we identified endothelial angiopoietin-2 (Ang-2) expression to be strongly associated with inflammations mediated by myeloid cells but not lymphocytes. To identify the underlying mechanism, we made use of a transgenic mouse model with inducible endothelial cell-specific expression of Ang-2. In this model, in the absence of inflammatory stimuli, long-term expression of Ang-2 led to a time-dependent accumulation of myeloid cells in numerous organs, suggesting that Ang-2 is sufficient to recruit myeloid cells. In models of acute inflammation, such as delayed-type hypersensitivity and peritonitis, Ang-2 transgenic animals showed an increased responsiveness. Intravital fluorescence video microscopy revealed augmented cell adhesion as an underlying event. Consequently, we demonstrated that Ang-2 is able to induce strong monocyte adhesion under shear in vitro, which could be blocked by antibodies to β2-integrin. Taken together, our results describe Ang-2 as a novel, endothelial-derived regulator of myeloid cell infiltration that modulates β2-integrin–mediated adhesion in a paracrine manner.

Background and purpose Gliomas display prime examples of ionizing radiation (IR) resistant tumors. The IAP Survivin is reported to be critically involved in radiation resistance by anti-apoptotic and by caspase-independent mechanisms. The present study aimed to elucidate an interrelationship between Survivin’s cellular localization and DNA damage repair in glioma cells. Material and methods Cellular distribution and nuclear complex formation were assayed by immunoblotting, immunofluorescence staining and co-immunoprecipitation of Survivin bound proteins in LN229 glioblastoma cells. Apoptosis induction, survival and DNA repair following IR were assayed by means of caspase3/7 activity, clonogenic assay, γ-H2AX/53BP1 foci formation, single cell gel electrophoresis assay, and DNA-PKcs kinase assay in the presence of Survivin siRNA or over expression of Survivin-GFP. Results Following irradiation, we observed a nuclear accumulation and a direct interrelationship between Survivin, MDC1, γ-H2AX, 53BP1 and DNA-PKcs, which was confirmed by immunofluorescence co-localization. Survivin downregulation by siRNA resulted in an increased apoptotic fraction, decreased clonogenic survival and increased DNA-damage, as demonstrated by higher amount of DNA breaks and an increased amount of γ-H2AX/53BP1 foci post irradiation. Furthermore, we detected in Survivin-depleted LN229 cells a hampered S2056 (auto)phosphorylation and a significantly decreased DNA-PKcs kinase activity. Conclusion Nuclear accumulation of Survivin and interaction with components of the DNA-double-strand break (DSB) repair machinery indicates Survivin to regulate DSB damage repair that leads to a significant improvement of survival of LN229 glioblastoma cells.

We recently reported that the vast majority (

Schittenhelm J, Simon P, Harter P N, Zachskorn C, Schlaszus H, Rottger F, Winkels M, Weller M, Meyermann R & Mittelbronn M (2011) Histopathology58, 739–749 CD133 expression is associated with small round blue cell tumour morphology in human central nervous system neoplasms Aims: CD133 is considered to be a marker for brain tumour-initiating cells. However, most data on CD133 are derived from animal or in-vitro studies. The aim of this study was to characterize CD133 expression, and the distribution and morphological features of CD133+ cells, in primary and secondary human central nervous system (CNS) neoplasms. Methods and results: Tumours were analysed by real-time reverse transcription polymerase chain reaction, western blot, flow cytometry and immunohistochemistry. Our results show that only small round blue cell tumours (SRBCTs) exhibit strong and consistent CD133 expression. Interestingly, glioblastomas, large-cell carcinomas and sarcomas were negative for CD133. Only glioblastomas with a focal small-cell component exhibited CD133 immunoreactivity in the SRBCT component. In addition, CD133 expression did not correlate with the expression of other markers associated with stem cell differentiation, including CD15 and nestin. Conclusions: CD133 expression in human CNS neoplasms is independent of the grade of malignancy but strongly correlates with SRBCT morphology. Together with recent findings showing that CD133 is quickly upregulated upon hypoxia and that CD133− cells can also exhibit stem cell properties, our data strongly question the suitability of CD133 as a brain tumour stem cell marker in vivo.

Medulloblastoma (MB) is the most common malignant brain tumor in children. It is known that overexpression and/or amplification of the MYC oncogene is associated with poor clinical outcome, but the molecular mechanisms and the MYC downstream effectors in MB remain still elusive. Besides contributing to elucidate how progression of MB takes place, most importantly, the identification of novel MYC-target genes will suggest novel candidates for targeted therapy in MB. A group of 209 MYC-responsive genes was obtained from a complementary DNA microarray analysis of a MB-derived cell line, following MYC overexpression and silencing. Among the MYC-responsive genes, we identified the members of the bone morphogenetic protein (BMP) signaling pathway, which have a crucial role during the development of the cerebellum. In particular, the gene BMP7 was identified as a direct target of MYC. A positive correlation between MYC and BMP7 expression was documented by analyzing two distinct sets of primary MB samples. Functional studies in vitro using a small-molecule inhibitor of the BMP/SMAD signaling pathway reproduced the effect of the small interfering RNA-mediated silencing of BMP7. Both approaches led to a block of proliferation in a panel of MB cells and to inhibition of SMAD phosphorylation. Altogether, our findings indicate that high MYC levels drive BMP7 overexpression, promoting cell survival in MB cells. This observation suggests the potential relevance of targeting the BMP/SMAD pathway as a novel therapeutic approach for the treatment of childhood MB.

The current World Health Organization classification recognizes three histological types of grade II low-grade diffuse glioma (diffuse astrocytoma, oligoastrocytoma, and oligodendroglioma). However, the diagnostic criteria, in particular for oligoastrocytoma, are highly subjective. The aim of our study was to establish genetic profiles for diffuse gliomas and to estimate their predictive impact. In this study, we screened 360 World Health Organization grade II gliomas for mutations in the IDH1, IDH2, and TP53 genes and for 1p/19q loss and correlated these with clinical outcome. Most tumors (86%) were characterized genetically by TP53 mutation plus IDH1/2 mutation (32%), 1p/19q loss plus IDH1/2 mutation (37%), or IDH1/2 mutation only (17%). TP53 mutations only or 1p/19q loss only was rare (2 and 3%, respectively). The median survival of patients with TP53 mutation ± IDH1/2 mutation was significantly shorter than that of patients with 1p/19q loss ± IDH1/2 mutation (51.8 months vs. 58.7 months, respectively; P = 0.0037). Multivariate analysis with adjustment for age and treatment confirmed these results (P = 0.0087) and also revealed that TP53 mutation is a significant prognostic marker for shorter survival (P = 0.0005) and 1p/19q loss for longer survival (P = 0.0002), while IDH1/2 mutations are not prognostic (P = 0.8737). The molecular classification on the basis of IDH1/2 mutation, TP53 mutation, and 1p/19q loss has power similar to histological classification and avoids the ambiguity inherent to the diagnosis of oligoastrocytoma.

Despite the blood–brain barrier (BBB) the human CNS is continuously screened by blood-derived immunological cells. In certain brain areas the local BBB configuration grants passage of large molecules, whereas others are better shielded. We investigated whether these regional BBB compositions are paralleled by differences in the degree of cellular immunosurveillance by investigating tissue from 23 normal human brains for several CD markers, FoxP3, granzyme B, and perforin. Our results provide evidence that immunosurveillance is associated with locoregional BBB configuration and is mainly performed by CD3+/CD8+/granzyme B-/perforin- lymphocytes.

The A kinase anchor protein 12 (AKAP12) is a central mediator of protein kinase A and protein kinase C signaling. Although AKAP12 has been described to act as a tumor suppressor and its expression is frequently down-regulated in several human malignancies, the underlying molecular mechanisms responsible for the AKAP12 reduction are poorly understood. We therefore analyzed the expression of AKAP12 and its genetic and epigenetic regulatory mechanisms in human hepatocarcinogenesis. Based on tissue microarray analyses (n = 388) and western immunoblotting, we observed a significant reduction of AKAP12 in cirrhotic liver (CL), premalignant lesions (DN), and hepatocellular carcinomas (HCCs) compared to histologically normal liver specimens (NL). Analyses of array comparative genomic hybridization data (aCGH) from human HCCs revealed chromosomal losses of AKAP12 in 36% of cases but suggested additional mechanisms underlying the observed reduction of AKAP12 expression in hepatocarcinogenesis. Quantitative methylation analysis by MassARRAY of NL, CL, DN, and HCC tissues, as well as of various tumorigenic and nontumorigenic liver cell lines revealed specific hypermethylation of the AKAP12α promoter but not of the AKAP12β promoter in HCC specimens and in HCC cell lines. Consequently, restoration experiments performed with 5-aza-2′deoxycytidine drastically increased AKAP12α mRNA levels in a HCC cell line (AKN1) paralleled by AKAP12α promoter demethylation. As hypermethylation is not observed in CL and DN, we investigated microRNA-mediated posttranscriptional regulation as an additional mechanism to explain reduced AKAP12 expression. We found that miR-183 and miR-186 are up-regulated in CL and DN and are able to target AKAP12. Conclusion: In addition to genetic alterations, epigenetic mechanisms are responsible for the reduction of the tumor suppressor gene AKAP12 in human hepatocarcinogenesis. (HEPATOLOGY 2010;.)

The question whether human cytomegalovirus may affect cancer diseases has been discussed (very controversially) for decades. There are convinced believers and strict opponents of the idea that HCMV might be able to play a role in the course of cancer diseases. In parallel, the number of published reports on the topic is growing. Recently published and presented (Ranganathan P, Clark P, Kuo JS, Salamat S, Kalejta RF. A Survey of Human Cytomegalovirus Genomic Loci Present in Glioblastoma Multiforme Tissue Samples. 35th Annual International Herpes Workshop, Salt Lake City, 2010) data on HCMV detection in glioblastoma tissues and colocalisation of HCMV proteins with cellular proteins known to be relevant for glioblastoma progression motivated us to recapitulate the current state of evidence.

Purpose: Growth and differentiation factor (GDF)-15 is a member of the transforming growth factor (TGF)-β family. GDF-15 is necessary for the maintenance of pregnancy but has also been linked to other physiologic and pathologic conditions. Experimental Design: The expression of GDF-15 in glioma cell lines was assessed by quantitative reverse transcriptase-PCR and immunoblot. GDF-15 levels in situ and in the peripheral blood of glioma patients were examined by immunohistochemistry and enzyme-linked immunosorbent assay, respectively. The effects of short hairpin RNA-mediated GDF-15 inhibition on proliferation and immunogenicity of SMA-560 glioma cells were investigated by [methyl-3H]thymidine incorporation and immune-mediated target cell lysis. The impact of GDF-15 on glioma growth in vivo was assessed in syngeneic mice. Results: GDF-15 is expressed by gliomas of different WHO grades as assessed by immunohistochemistry. The high expression of GDF-15 in tumor tissue translates into elevated GDF-15 serum levels in glioblastoma patients compared with healthy controls. GDF-15 mRNA and protein are also detectable in human and mouse glioma cells in vitro. Silencing of GDF-15 by RNA interference reduces the proliferation of malignant glioma cells. Immunologically, the depletion of glioma-derived GDF-15 enhances the susceptibility of mouse glioma cells towards syngeneic natural killer cells and splenocytes. This results in a reduced in vivo tumorigenicity and increased T-cell infiltration of GDF-15–deficient glioma cells in syngeneic mice. Conclusions: Although previous studies focusing on ectopic overexpression of GDF-15 have proposed unclear or antitumorigenic effects of GDF-15 in glioma cells, we here show that GDF-15 at endogenous levels contributes to proliferation and immune escape of malignant gliomas in an immunocompetent host. Clin Cancer Res; 16(15); 3851–9. ©2010 AACR.

P. N. Harter, B. Bunz, K. Dietz, K. Hoffmann, R. Meyermann and M. Mittelbronn (2010) Neuropathology and Applied Neurobiology36, 623–635 Spatio-temporal deleted in colorectal cancer (DCC) and netrin-1 expression in human foetal brain development Aims: Deleted in colorectal cancer (DCC) and its ligand netrin-1 are known as axonal guidance factors, being involved in angiogenesis, migration and survival of precursor cells in the embryonic mammalian central nervous system (CNS). So far, little is known about the distribution of those molecules in human CNS development. Methods: We investigated 22 human foetal brain specimens (12th and 28th week of gestation) for DCC and netrin-1 expression by means of immunohistochemistry, immunofluorescence and confocal laser microscopy. Statistical analysis was performed by applying a semi-quantitative score, including staining intensity and frequency and correlation with foetal age. Results: DCC and netrin-1 were differentially expressed throughout the developing human foetal telencephalic and cerebellar cortical layers. Netrin-1 exhibited the highest levels in telencephalic germinal layers, whereas the strongest DCC immunoreactivity was seen in the developing cortical plate. Netrin-1 and DCC were predominantly present on cerebellar external granule layer cells. Distinct co-expression was seen in maturing foetal brainstem nuclei, cerebellar external granular layer and the choroid plexus. In contrast, endothelial cells showed strong netrin-1 expression with subsidiary DCC immunoreactivity. Pontine and telencephalic axonal fibre tracts also demonstrated strong netrin-1 expression. Conclusions: We show that DCC and netrin-1 are ubiquitously expressed in the human foetal brain; however, both exhibit a distinct spatio-temporal expression pattern. Together with the data from animal experiments, our findings might indicate also an important role for DCC and netrin-1 in human foetal CNS development.

Up to 8% of patients with gluten sensitivity (GS) develop neurological symptoms such as ataxia, dementia, seizures or peripheral neuropathy. The underlying immunological mechanisms still remain to be elucidated. We here report the case of a 68-year-old male patient suffering from progressive ataxia and dementia associated with chronic diarrhea and both elevated IgG and IgA antigliadin-antibodies. At autopsy, frequent argyrophilic glial and neuronal inclusions within the basal nucleus of Meynert were considered as the structural correlative for the cognitive decline. Significant neuronal loss in the cerebellar cortex and the inferior olives was accompanied by infiltrating CD8(+)/perforin(+)/granzyme B(+) cells as well as reactive astrogliosis and microglial activation. These CD8(+) cytotoxic T and NK cells are likely to act as effector cells responsible for neuronal cell death in patients with gluten sensitivity and neurological disease and might therefore at least partly be responsible for cerebellar symptoms in gluten ataxia. In conclusion, our results, showing an absence of B- or plasma cells but multiple CD8(+) as well as granzyme B and perforin expressing cells in ataxia-associated brain areas, suggest that there are also prominent cytotoxic effects in neuropathogenesis of GS.

INTRODUCTION Glioblastoma multiforme remains one of the most difficult to treat cancers due to the highly infiltrative nature of cancer cells. Targeting the invasive properties of glioblastoma presents a novel treatment avenue. Recently, specific GSK-3 beta (GSK3b) inhibitors such as Bio-Indirubin (BIO), have been described that inhibit the migratory activity of glioma cells. In this study, the GSK-3b downstream targets were investigated.

High biological activity of the transforming growth factor (TGF)-beta-Smad pathway characterizes the malignant phenotype of malignant gliomas and confers poor prognosis to glioma patients. Accordingly, TGF-beta has become a novel target for the experimental treatment of these tumors. TGF-beta is processed by furin-like proteases (FLP) and secreted from cells in a latent complex with its processed propeptide, the latency-associated peptide (LAP). Latent TGF-beta-binding protein 1 (LTBP-1) covalently binds to this small latent TGF-beta complex (SLC) and regulates its function, presumably via interaction with the extracellular matrix (ECM). We report here that the levels of LTBP-1 protein in vivo increase with the grade of malignancy in gliomas. LTBP-1 is associated with the ECM as well as secreted into the medium in cultured malignant glioma cells. The release of LTBP-1 into the medium is decreased by the inhibition of FLP activity. Gene-transfer mediated overexpression of LTBP-1 in glioma cell lines results in an increase inTGF-beta activity. Accordingly, Smad2 phosphorylation as an intracellular marker of TGF-beta activity is enhanced. Conversely, LTBP-1 gene silencing reduces TGF-beta activity and Smad2 phosphorylation without affecting TGF-beta protein levels. Collectively, we identify LTBP-1 as an important modulator of TGF-beta activation in glioma cells, which may contribute to the malignant phenotype of these tumors.

Sporadic inclusion body myositis (sIBM) is a debilitating idiopathic inflammatory myopathy. Little is known about the pathogenetic mechanisms that lead to myofiber degeneration. In the present study, we evaluated the integrity of the myofiber basement membrane in non-necrotic myofibers invaded by inflammatory infiltrates. We used 100 ten mum thick serial sections obtained from biopsies of 5 patients suffering from sIBM. Biopsies from 5 patients suffering from polymyositis served as controls. We performed sequential HE staining and immunolabeling using anti-CD68, -CD8, -merosin, -laminin alpha4 chain, and -collagen IV antibodies. In sIBM, we detected a total of 89 non-necrotic myofibers that were invaded by inflammatory cells. The invasive process and its sequelae were segmental in nature and included destruction of the myofiber basement membrane, and eventually, partial replacement by fibrosis of the invaded myofiber. In polymyositis, we found only two myofibers that were affected in this way. In sIBM, basement membrane remodelling and irreversible replacement by fibrosis of myofibers appear to represent the end result of a process in which the balance between injury and repair are disrupted.

Microscopic distinction of normal choroid plexus (CP) from choroid plexus tumors (CPT) may be difficult, especially in small samples of well-differentiated CP papillomas. So far, there are no established markers that reliably distinguish normal and neoplastic CP epithelium. Recently, a correlation between expression/function of glial glutamate transporters EAAT-1 (GLAST) and EAAT-2 (Glt-1) and tumor proliferation has been reported. Furthermore, we previously found that CPTs frequently express EAAT-1, but not EAAT-2. We now compared expression of EAAT-1, EAAT-2 and GFAP in non-neoplastic CP (n = 68) and CPT (n = 79) by immunohistochemistry. Tissue of normal CP was obtained from 50 autopsy cases (20 normal and 30 pathologic brains) and 18 neurosurgical specimens that included 17 fetal, 21 pediatric and 30 adult cases. In non-neoplastic postnatal CP (n = 51), focal expression of EAAT-1 was found in only two pediatric cases (4%). In CPT, expression of EAAT-1 was found in 64 of 79 (81%) tumor samples and was significantly age-dependent (P < 0.0001). Hence, EAAT-1 expression distinguishes neoplastic from normal CP, both in children (P = 0.0032) and in adults (P < 0.0001). Immunostaining for EAAT-2 in selected samples from cases of different ages showed that normal CP (n = 15) or CPT (n = 16) lacked EAAT-2 expression. GFAP expression was found in 3 of 32 (10%) normal CP and in 28 of 73 (38%) tumor samples. In conclusion, in contrast to neoplastic CP samples, expression of EAAT-1 is exceptionally rare in non-neoplastic CP. Thus, EAAT-1 is superior to GFAP as a helpful diagnostic tool in CP samples.

Aims: The Epo-EpoR pathway plays a role in tumour growth, metastasis and treatment resistance and is a potential target in oncological treatment. As the EpoR status in human meningiomas is unknown, our aim was to characterize EpoR expression in these tumours. Methods: We examined 131 meningioma samples of all WHO grades from 116 patients by immunohistochemistry for EpoR. Among these, 25 meningiomas showed brain invasion and 29 patients had a further tumour recurrence. A group of 20 patients without tumour recurrence served as controls. In 12 cases we were able to compare both the primary and the following recurrent tumours. The presence of EpoR in meningiomas was confirmed by RT-PCR and Western blot. Results: EpoR was expressed in all meningiomas. Statistical analysis revealed that the mean expression levels of EpoR were significantly lower in primary tumours with known recurrence compared with a recurrence-free control group. Additional matched pair analysis in individual cases showed no significant differences between primary and recurrent tumours. No significant correlation between EpoR expression and WHO grade, age, sex or brain invasion was detected. Using specific primer pairs for RT-PCR, we were able to detect all three known isoforms of EpoR: the full-length isoform EpoR-F, the truncated isoform EpoR-T and the soluble isoform EpoR-S. Conclusions: Our results demonstrate the expression of EpoR in meningiomas. Lower EpoR mean levels might be a useful marker for a higher recurrence risk, but further studies are needed to clarify the influence of EpoR on recurrences and the role of the different isoforms.

O6-methylguanine-DNA methyltransferase (MGMT) is a repair enzyme that removes promutagenic O6-methylguanine adducts in DNA, to protect cells from acquisition of G:C→ A:T mutations. MGMT promoter methylation and polymorphisms may affect MGMT expression and activity. In the present study, we assessed MGMT promoter methylation and polymorphisms (Leu84Phe, Ile143Val, c.–56C>T) in 371 glioblastomas diagnosed at the population level. MGMT methylation was observed in 165 (44%) glioblastomas, with a higher frequency in females than males (53 vs. 39%; p = 0.0106) and in secondary than primary glioblastomas (73 vs. 43%; p = 0.0074). The frequency of TP53 G:C→A:T mutations in glioblastomas with MGMT methylation was 25%, which was significantly higher than that in glioblastomas with MGMT methylation (16%; Fisher exact test; p = 0.0385). MGMT 143 Val allele in glioblastomas was significantly less frequent than in a healthy European Caucasian population, and was associated with longer survival than those with the MGMT 143 Ile allele (hazard ratio 0.70; 95% CI 0.48–1.01). These results suggest that MGMT methylation may be associated with susceptibility to acquire TP53 G:C→A:T mutations, and that MGMT polymorphisms may affect the risk and prognosis of glioblastomas.

Exencephaly/anencephaly is a rare neural tube defect occurring early in embryogenesis. We report a 14-week-old fetus with exencephaly in whom central nervous system tissue was developed and preserved. There were 2 symmetrical structures grossly resembling cerebral hemispheres, which on histologic and ultrastructural study, consisted of a combination of ependymoblastomatous rosettes and canals and primitive neural tissue. The brainstem and spinal cord were partially normally formed, although descending tracts were not apparent. No cerebellar tissue was found. The eyes were formed. This appears to represent a rare example of exencephaly not covered by skin, which did not undergo necrosis and early transformation into a residual area cerebrovasculosa, characteristic of anencephaly. It may be appropriate to regard this as a unique neural tube closure defect that might be termed “ependymoblastomatous exencephaly.”

Haematopoietic progenitor cells (HPC) are attracted by experimental gliomas in vivo. This attraction is further enhanced by irradiation or hypoxic preconditioning of the glioma cells. Adhesive interactions might be critical to the preferential accumulation of HPC within the glioma tissue. Here, we studied the interactions of HPC with endothelial cells. Exposure of human cerebral endothelial cells (SV-HCEC), human microvascular endothelial cells (HMEC) and brain tumour endothelial cells derived from human glioblastomas (BTEC) to supernatants of glioma cells and primary glioma cells (SN-G) induced the expression of E-selectin (CD62E). CD62E expression was further enhanced when the glioma cells had been exposed to irradiation or hypoxia prior to the collection of supernatants, as well as by irradiation or exposure to hypoxia of the endothelial cells. Vascular cell adhesion molecule 1 (VCAM-1) was constitutively expressed on SV-HCEC, HMEC and BTEC, but was not modulated by SN-G, irradiation or hypoxia. Transendothelial HPC migration was enhanced after CD62E induction in vitro. Neutralizing antibodies to CD62E strongly reduced the homing of lin(-)Sca-1(+)c-kit(+) cells to orthotopic SMA-560 gliomas in vivo. Tissue microarray sampling normal brain tissue and astrocytomas of WHO grades II-IV revealed a selective expression of CD62E on endothelial cells of tumour vessels. SN-G-induced CD62E expression on endothelial cells in vitro required transforming growth factor (TGF)-beta signalling in glioma cells and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGF-R2) signalling in endothelial cells. Further, we observed a nuclear factor kappa B-dependent activation of the CD62E promoter peaking at 12 h after VEGF-R2 activation by glioma-derived VEGF. Taken together, we identify glioma cell-induced CD62E expression on endothelial cells as one mediator of the glioma tropism of HPC.

The co-inhibitory B7-homologue 1 (B7-H1/PD-L1) influences adaptive immune responses and has been proposed to contribute to the mechanisms maintaining peripheral tolerance and limiting inflammatory damage in parenchymal organs. To understand the B7-H1/PD1 pathway in CNS inflammation, we analyzed adaptive immune responses in myelin oligodendrocyte glycoprotein (MOG)(35-55)-induced EAE and assessed the expression of B7-H1 in human CNS tissue. B7-H1(-/-) mice exhibited an accelerated disease onset and significantly exacerbated EAE severity, although absence of B7-H1 had no influence on MOG antibody production. Peripheral MOG-specific IFN-gamma/IL-17 T cell responses occurred earlier and enhanced in B7-H1(-/-) mice, but ceased more rapidly. In the CNS, however, significantly higher numbers of activated neuroantigen-specific T cells persisted during all stages of EAE. Experiments showing a direct inhibitory role of APC-derived B7-H1 on the activation of MOG-specific effector cells support the assumption that parenchymal B7-H1 is pivotal for delineating T cell fate in the target organ. Compatible with this concept, our data investigating human brain tissue specimens show a strong up-regulation of B7-H1 in lesions of multiple sclerosis. Our findings demonstrate the critical importance of B7-H1 as an immune-inhibitory molecule capable of down-regulating T cell responses thus contributing to the confinement of immunopathological damage.

ObjectiveRecent observational studies have established progression and recurrence rates of pituitary adenomas. However, it is still unknown how individual pituitary adenomas grow over years and whether growth kinetics follow a distinct growth model. The objective of this study was to define a growth model for non-functioning pituitary adenomas.MethodsFifteen patients who had five or more serial high-quality examinations with magnetic resonance images or computerized tomography scans were identified among 216 patients with non-functioning pituitary adenomas. Tumour volumes were assessed using a stereological method based on the Cavalieri principle. Tumour growth during the observation period was analysed and different growth models were fitted to the data.ResultsFifteen pituitary adenomas (12 recurrent tumours and 3 newly diagnosed tumours) were longitudinally observed during a median observation period of 7.4 years (range: 2.3–11.9 years). Growth kinetics could be described either by an exponential growth model (nine patients) or by a logistic model (five patients) with initial exponential growth followed by deceleration of growth. One tumour remained unchanged in size during the observation period. None of the adenomas showed accelerated growth during the observation period. Overall, the linear growth model was not suitable to describe the growth kinetics of non-functioning pituitary adenomas.ConclusionsOur study shows that growth of pituitary adenomas can be described by distinct growth models. Knowledge of growth dynamics has implications for clinical practice and helps to adjust scanning protocols for follow-up investigations.

[(11)C]PIB ((11)C-6-OH benzothiazole) reflects the regional distribution of amyloid (beta-sheeted proteins) in patients with Alzheimer's disease (AD). Proteinaceous inclusions in Parkinson's disease with dementia (PDD), so-called Lewy bodies, also consist of fibrillar, misfolded proteins, chiefly alpha-synuclein. To test whether PDD subjects show specific amyloid binding in vivo and whether this could reflect fibrillar alpha-synuclein accumulation, we investigated 10 PDD subjects with [(11)C]PIB-PET. Radioligand binding was compared to that in 11 control and 6 AD subjects. Furthermore, postmortem sections of 4 patients with Parkinson's disease (PD), therefrom 2 with dementia (PDD), and of 6 controls were stained with PIB to evaluate the histological distribution of the fluorescent ligand in the brainstem. In PET, only 2 PDD patients displayed increased PIB binding to cortical amyloid comparable to AD patients. The other 8 patients showed control-like cortical findings but elevated PIB binding in the pons and mesencephalon. Fluorescence microscopy showed PIB binding to Lewy bodies and neuromelanin in the substantia nigra of PD and PDD brainstem sections, but not in controls. These data suggest that PIB-PET can be used to further differentiate PDD with respect to cortical amyloid. Furthermore, we provide evidence that--in addition to nonspecific binding--PIB uptake in the brainstem may also reflect PDD related amyloid.

Immunohistochemical studies showed that O6-methylguanine-DNA methyltransferase (MGMT) protein expression is negatively associated with survival in glioblastomas treated with alkylating agents in accordance with previous results of methylation-specific PCR. Implementation of this data in routine clinical diagnostics is limited due to often inappropriate study designs, e.g. pooling of tumor entities, WHO grades or primary and secondary glioblastomas, disregard concerning the infiltration zone or various epidemiological factors. The aim of our study was to evaluate MGMT expression and its prognostic value taking into consideration the aforementioned deficiencies. For this, 162 astrocytic tumors WHO II–IV (36 diffuse astrocytomas WHO II, 51 anaplastic astrocytomas, 75 primary glioblastomas) as well as 25 glioblastoma infiltration zones and 19 glioblastoma relapses were analyzed for immunohistochemical MGMT protein expression using tissue microarray technique. Expression of MGMT significantly decreased from WHO grade II (25.6%) to glioblastoma (16.8%, p = 0.01) with lowest levels in grade III tumors (10.2%, II/III p

Background— Extracellular adenosine, generated from extracellular nucleotides via ectonucleotidases, binds to specific receptors and provides cardioprotection from ischemia and reperfusion. In the present study, we studied ecto-enzymatic ATP/ADP-phosphohydrolysis by select members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family during myocardial ischemia. Methods and Results— As a first step, we used a murine model of myocardial ischemia and in situ preconditioning and performed pharmacological studies with polyoxometalate 1, a potent E-NTPDase inhibitor (Na 6 [H 2 W 12 O 40 ]). Polyoxometalate 1 treatment increased infarct sizes and abolished beneficial effects of preconditioning. To define relative contributions of distinct E-NTPDases, we investigated transcriptional responses of E-NTPDases 1 to 3 and 8 to preconditioning. We noted robust and selective induction of E-NTPDase 1 (CD39) transcript and protein. Histological analysis of preconditioned myocardium localized CD39 induction to endothelia and myocytes. Cd39 −/− mice exhibited larger infarct sizes with ischemia ( cd39 +/+ 43.0±3.3% versus cd39 −/− 52%±1.8; P cd39 +/+ 13.3%±1.5 versus cd39 −/− 50.5%±2.8; P cd39 −/− mice were corrected by infusion of the metabolic product (AMP) or apyrase. Moreover, apyrase treatment of wild-type mice resulted in 43±4.2% infarct size reduction ( P Conclusions— Taken together, these studies reveal E-NTPDase 1 in cardioprotection and suggest apyrase in the treatment of myocardial ischemia.

Gangliogliomas are rare CNS neoplasms mostly occuring in young adults and are usually assigned to WHO grade I. The few cases of WHO grade IV gangliogliomas were either primarily malignant glio-neuronal tumors or underwent malignant progression from other WHO grades after radiotherapy. Herein, we present the case of a now 47-year-old female patient presenting with a benign ganglioglioma and showing a tumor recurrence 2 years later with an anaplastic ganglioglioma, assigned to WHO grade IV, with malignant transformation in both glial and neuronal components. The presented case is the first reported low-grade ganglioglioma rapidly progressing to a WHO grade IV glio-neuronal tumor not being associated with radiotherapy and showing malignant transformation in both astroglial and neuronal tumor cell components.

We report the case of a multifocal dysembryoplastic neuroepithelial tumor (DNT) in a 7-year-old girl with local tumor regrowth 6 years later. The tumor was localized in the right parietal lobe extending from the cortex into the periventricular white matter. After subtotal resection of a histopathologically confirmed DNT we observed unexpected tumor progression in long-term follow-up. Therefore, a second surgery was performed when the patient was 14 years of age. In neuropathological examination of the second specimen the tumor showed an increased cellularity and pleomorphism, microvascular proliferations, an elevated proliferative activity (MIB1-index focally up to 10%) and cellular atypia not typical for WHO grade I DNT. Furthermore, MRI studies showed additional supratentorial and infratentorial lesions which remained stable over years and are also well consistent with DNTs. Thus, an unusual form of a DNT with multifocal lesions, local regrowth and morphological transformation is supposed.

Acute lung injury (ALI), such as that which occurs with mechanical ventilation, contributes to morbidity and mortality of critical illness. Nonetheless, in many instances, ALI resolves spontaneously through unknown mechanisms. Therefore, we hypothesized the presence of innate adaptive pathways to protect the lungs during mechanical ventilation. In this study, we used ventilator-induced lung injury as a model to identify endogenous mechanisms of lung protection. Initial in vitro studies revealed that supernatants from stretch-induced injury contained a stable factor which diminished endothelial leakage. This factor was subsequently identified as adenosine. Additional studies in vivo revealed prominent increases in pulmonary adenosine levels with mechanical ventilation. Because ectoapyrase (CD39) and ecto-5′-nucleotidase (CD73) are rate limiting for extracellular adenosine generation, we examined their contribution to ALI. In fact, both pulmonary CD39 and CD73 are induced by mechanical ventilation. Moreover, we observed pressure- and time-dependent increases in pulmonary edema and inflammation in ventilated cd39−/− mice. Similarly, pharmacological inhibition or targeted gene deletion of cd73 was associated with increased symptom severity of ventilator-induced ALI. Reconstitution of cd39−/− or cd73−/− mice with soluble apyrase or 5′-nucleotidase, respectively, reversed such increases. In addition, ALI was significantly attenuated and survival improved after i.p. treatment of wild-type mice with soluble apyrase or 5′-nucleotidase. Taken together, these data reveal a previously unrecognized role for CD39 and CD73 in lung protection and suggest treatment with their soluble compounds as a therapeutic strategy for noninfectious ALI.

✓Inflammatory myofibroblastic tumors with involvement of cranial and peripheral nerves are exceedingly rare. The authors present the case of a 67-year-old man with an inflammatory myofibroblastic tumor of the left ulnar nerve, which was identified intraoperatively and mimicked a malignant neoplastic lesion. Histopathological examination revealed loosely structured fibrous tissue and collagen deposits intermingled with patchy infiltrates of lymphocytes, plasma cells, and histiocytes penetrating the endo- and epineurium of the affected nerve fascicles. There was strong expression of vimentin and actin in spindle cells throughout the lesion. The histiocytes were CD68- and major histocompatibility complex class II–positive, but lacked CD1a expression. A review of the literature revealed nine histopathologically confirmed cases of inflammatory myofibroblastic tumors involving peripheral or cranial nerves in which slight differences in histopathological features and surgical management were found, which are discussed here.

Aims: Glutamate receptor antagonists have failed clinical stroke trials and it has been proposed that the action of N-methyl d-aspartate receptors is necessary for neuronal survival. Thus, excitatory amino acid transporters (EAATs) might be a promising therapeutic target. The aim of this study was to investigate glial expression of EAATs following ischaemia. Methods and results: Expression of EAAT1 (GLAST) and EAAT2 (Glt-1) in 24 cases of ischaemia was examined by immunohistochemistry. Cortical expression of both EAATs in the lesion decreased within 24 h (P

Defects of major histocompatibility complex (MHC) class I antigen-processing machinery (APM) components have been shown to contribute to immune escape of malignant cells. We investigated the expression of APM components in astrocytomas without detectable defects in HLA class I antigen expression and correlated it with grade of malignancy. Quantitative immunohistochemical analysis of astrocytomas revealed reduced expression of the cytosolic proteasome subunit low molecular weight protein 2 (LMP2), the endoplasmatic reticulum (ER) transporter associated with antigen processing-1 (TAP1), and the ER chaperone beta2-microglobulin (beta2m) in astrocytoma cells when compared to astrocytes from nonpathological brain. Among human WHO grade II-IV astrocytomas, downregulation of LMP2, TAP1 and beta2m correlated with grade of malignancy. Furthermore, astrocytoma cell lines (n = 12) expressed all APM components analyzed at levels comparable to dendritic cells (DC), which were used for comparative purposes. However, upregulation of beta2m after stimulation with inflammatory cytokines was significantly lower in astrocytoma cell lines than in control cells. Our results support the hypothesis that coordinated downregulation or impaired upregulation of certain HLA class I APM components may serve as a mechanism for astrocytoma cells to evade the host's immune response, even if HLA class I antigen surface expression is not altered.