Intratumoural immune cell landscape in germinoma reveals multipotent lineages and exhibits prognostic significance.

AIMS: Alterations in microenvironments are a hallmark of cancer, and these alterations in germinomas are of particular significance. Germinoma, the most common subtype of central nervous system germ cell tumours, often exhibits massive immune cell infiltration intermingled with tumour cells. The role of these immune cells in germinoma, however, remains unknown. METHODS: We investigated the cellular constituents of immune microenvironments and their clinical impacts on prognosis in 100 germinoma cases. RESULTS: Patients with germinomas lower in tumour cell content (i.e. higher immune cell infiltration) had a significantly longer progression-free survival time than those with higher tumour cell contents (P = 0.03). Transcriptome analyses and RNA in-situ hybridization indicated that infiltrating immune cells comprised a wide variety of cell types, including lymphocytes and myelocyte-lineage cells. High expression of CD4 was significantly associated with good prognosis, whereas elevated nitric oxide synthase 2 was associated with poor prognosis. PD1 (PDCD1) was expressed by immune cells present in most germinomas (93.8%), and PD-L1 (CD274) expression was found in tumour cells in the majority of germinomas examined (73.5%). CONCLUSIONS: The collective data strongly suggest that infiltrating immune cells play an important role in predicting treatment response. Further investigation should lead to additional categorization of germinoma to safely reduce treatment intensity depending on tumour/immune cell balance and to develop possible future immunotherapies.

Integrin α3 is overexpressed in glioma stem-like cells and promotes invasion.

BACKGROUND: Glioma stem-like cell (GSC) properties are responsible for gliomagenesis and recurrence. GSCs are invasive but its mechanism remains to be elucidated. Here, we attempted to identify the molecules that promote invasion in GSCs. METHODS: Neurospheres and CD133⁺ cells were collected from glioblastoma (GBM) specimens and glioma cell lines by sphere-formation method and magnetic affinity cell sorting, respectively. Differential expression of gene candidates, its role in invasion and its signaling pathway were evaluated in glioma cell lines. RESULTS: Neurospheres from surgical specimens attached to fibronectin and laminin, the receptors of which belong to the integrin family. Integrin α3 was overexpressed in CD133⁺ cells compared with CD133⁻ cells in all the glioma cell lines (4 out of 4). Immunohistochemistry demonstrated the localisation of integrin α3 in GBM cells, including invading cells, and in the tumour cells around the vessels, which is believed to be a stem cell niche. The expression of integrin α3 was correlated with migration and invasion. The invasion activity of glioma cells was linked to the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. CONCLUSION: Our results suggest that integrin α3 contributes to the invasive nature of GSCs via ERK1/2, which renders integrin α3 a prime candidate for anti-invasion therapy for GBM.

Visualization of angiographical arteriovenous shunting in perisylvian glioblastomas.

BACKGROUND: Arteriovenous shunting visualized by angiography is one of the major features of glioblastomas, and the visualization is dependent on the presence of extensive shunting. Extensive arteriovenous shunting is associated with the risk of poorly controlled intraoperative bleeding. When a tumor with extensive arteriovenous shunting is located in close proximity to the eloquent regions of the brain, a meticulous surgical procedure is necessary. In the present study, the site-oriented visualization of angiographical arteriovenous shunting was evaluated from the perspective of surgical treatment, with a particular focus on the perisylvian region that is in close proximity to motor and language regions (dominant hemisphere), as well as large arteries and veins. METHODS: Twenty-six consecutive patients underwent a resection of glioblastoma between February 2007 and September 2012. All patients were presurgically examined using digital subtraction angiography. The patients were subdivided into the following two groups based on the location of the tumor: 1) perisylvian glioblastoma (18 patients) and 2) non-perisylvian glioblastoma (eight patients). Angiography to detect the arteriovenous shunting was performed. In addition, the number of intratumoral vessels, tumor proliferative activity (MIB-1 labeling index), and volume of intraoperative bleeding were evaluated and compared between the two groups. RESULTS: Angiographical arteriovenous shunting was definitively visualized in 13 of 18 (72 %) perisylvian glioblastomas, in contrast to only one of eight (13 %) non-perisylvian glioblastomas (p = 0.007). There were no significant differences between the two groups with respect to the number of intratumoral vessels, MIB-1 labeling index, and volume of intraoperative bleeding. However, massive intraoperative bleeding of > 2,000 mL occurred in one perisylvian glioblastoma patient. CONCLUSIONS: Glioblastomas in the perisylvian region tend to be associated with extensive arteriovenous shunting that can be definitively visualized by performing an angiography. Because arteriovenous shunting carries the risk of intraoperative bleeding, perisylvian glioblastomas-particularly in the dominant hemisphere-should be resected with a meticulous surgical procedure and strategy.

Silencing of ferrochelatase enhances 5-aminolevulinic acid-based fluorescence and photodynamic therapy efficacy.

BACKGROUND: Recurrence of glioma frequently occurs within the marginal area of the surgical cavity due to invading residual cells. 5-Aminolevulinic acid (5-ALA) fluorescence-guided resection has been used as effective therapeutic modalities to improve discrimination of brain tumour margins and patient prognosis. However, the marginal areas of glioma usually show vague fluorescence, which makes tumour identification difficult, and the applicability of 5-ALA-based photodynamic therapy (PDT) is hampered by insufficient therapeutic efficacy in glioma tissues. METHODS: To overcome these issues, we assessed the expression of ferrochelatase (FECH) gene, which encodes a key enzyme that catalyses the conversion of protoporphyrin IX (PpIX) to heme, in glioma surgical specimens and manipulated FECH in human glioma cell lines. RESULTS: Prominent downregulation of FECH mRNA expression was found in glioblastoma tissues compared with normal brain tissues, suggesting that FECH is responsible for PpIX accumulation in glioblastoma cells. Depletion of FECH by small interference RNA enhanced PpIX fluorescence after exposure to 5-ALA concomitant with increased intracellular PpIX accumulation in glioma cells. Silencing of FECH caused marked growth inhibition and apoptosis induction by PDT in glioma cells. CONCLUSION: These results suggest that knockdown of FECH is a potential approach to enhance PpIX fluorescent quality for optimising the subjective discrimination of vague fluorescence and improving the effect of 5-ALA-PDT.

Neonatal exposure to thymotropic gross murine leukemia virus induces virus-specific immunologic nonresponsiveness.

Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses.

Identification of Vortex Cores in Cerebral Aneurysms on 4D Flow MRI.

BACKGROUND AND PURPOSE: The complexity and instability of the vortex flow in aneurysms are factors related to the rupture risk of unruptured cerebral aneurysms. We identified aneurysm vortex cores on 4D flow MR imaging and examined the relationship of these factors with the characteristics of cerebral aneurysms. MATERIALS AND METHODS: We subjected 40 aneurysms (37 unruptured, 3 ruptured) to 4D flow MR imaging. We visualized streamlines with velocities below the threshold-that is, a percentage value of the aneurysm maximum inflow velocity-and progressively decreased the threshold to identify vortex cores as thin, streamline bundles with minimum velocities. Complexity and stability were compared in aneurysms with a smooth surface and those with blebs or daughter sacs. RESULTS: The threshold for visualizing vortex cores ranged from 3% to 13% of the maximum inflow velocity. Vortex cores could be visualized in 38 aneurysms; in 2, they were not visualized through the cardiac cycle. A simple flow pattern (single vortex core) was identified in 27 aneurysms; the other 13 exhibited a complex flow pattern. The cores were stable in 32 and unstable in 8 aneurysms. Significantly more aneurysms with-than-without blebs or daughter sacs had a complex flow pattern (P = .006). Of the 3 ruptured aneurysms, 1 aneurysm had an unstable vortex core; in the other 2, the vortex core was not visualized. CONCLUSIONS: The identification of vortex cores on 4D flow MR imaging may help to stratify the rupture risk of unruptured cerebral aneurysms.

A clinical study assessing the tolerability and biological effects of infliximab, a TNF-alpha inhibitor, in patients with advanced cancer.

BACKGROUND: Tumour necrosis factor-alpha (TNF-alpha) is an important regulator of the chronic inflammation contributing to tumour progression. Infliximab, an anti-TNF-alpha monoclonal antibody was investigated in this trial of patients with advanced cancer. The primary objectives were to determine the safety profile and biological response of infliximab in a cancer population. Clinical response was a secondary objective. PATIENTS AND METHODS: Forty-one patients received infliximab at 5 mg/kg (n = 21) or 10 mg/kg (n = 20) i.v. at 0 and 2 weeks and then every 4 weeks. Post-treatment samples were measured for changes in plasma and serum TNF-alpha, CCL2, IL-6 and C-reactive protein (CRP). RESULTS: Infliximab was well tolerated with no dose-limiting toxic effects. At both doses of infliximab, neutralisation of serum TNF-alpha was observed after 1 h while plasma CCL2, IL-6 and serum CRP were decreased 24 and 48 h following infliximab administration. Seven patients experienced disease stablisation (range 10-50+ weeks). There was no evidence of disease acceleration in any patient. CONCLUSIONS: Infliximab treatment was safe and well tolerated in patients with advanced cancer. There was evidence of biological activity with baseline TNF-alpha and CCL2 being correlated with infliximab response.

Optical properties of pb(Zr,Ti)O(3) films prepared by aerosol deposition.

The optical properties and related band structure of ferroelectric lead zirconate titanate [PZT, Pb(Zr0(0.6)Ti0(0.4))O(3)] films prepared on glass substrates at room temperature by aerosol deposition were investigated. The reflectance and transmittance of the PZT films were measured in the wavelength range from UV to nearinfrared. The measured optical spectra were analyzed using dielectric function models that describe optical transitions in the band gap region. Optical absorption of the as-deposited PZT films was found to be larger than that of the annealed PZT films in the near-infrared wavelength range. The analyzed results indicated that post-deposition annealing increased the band gap energy of the PZT films, corresponding to a decrease in optical absorption.

The characteristics of simple muscle power by gripping: gender differences and reliability of parameters using various loads.

AIM: There are few studies on muscle power during local muscle contractions with a small range of motion such as in gripping. The purposes of this study were to clarify the properties of the developmental phase based on time series of muscle power output, the reliability of the parameters, their relationships and the load intensity derived peak power by gender differences, and to examine the possibility of evaluating muscle power using gripping. METHODS: Fifteen young males and 15 females participated in this study. Based on a crossover experimental design, each subject carried out 2 explosive grips at 20%, 30%, 40% and 50% loads of maximal using a voluntary grip contraction (MVC). The grip contraction velocities, sampled at 100 Hz, were measured accurately using a power instrument with an accelerometer. Muscle power curves were drawn from the product of the velocity and the set-up load. RESULTS: The cross-correlation coefficients between the trials for the average curve of the time-series moving distance, the velocity, and the power in any load were very high (over 0.95) in both genders. The reliability of each parameter was mostly good in both genders (intraclass correlation coefficient, ICC>0.75). The peak power curve differed between genders, and the curve around the peak value in females was irregular. CONCLUSIONS: A gender difference was found in the maximal power and the properties of the power curve. The maximal muscle power appeared at 30-50% MVC in males, and at 20-40% MVC in females. The peak power during the whole contraction, and the time to peak may reflect the conditions throughout the whole of the contraction. The new device used in this study to evaluate local regional muscle power (grip) is a very reliable and useful tool.

Expression of dominant-negative form of Ets-1 suppresses fibronectin-stimulated cell adhesion and migration through down-regulation of integrin alpha5 expression in U251 glioma cell line.

Ets transcription factors are associated with tumor malignancy. We reported previously that the stable transfection of the dominant-negative form of Ets-1 (Ets-DN) in the glioma cell line U251 induced down-regulation of urokinase-type plasminogen activator mRNA expression and invasiveness (M. Nakada et al., J. Neuropathol. Exp. Neurol., 58: 329-334, 1999). Here we analyzed effects of Ets-DN expression on cell adhesion, migration, and phosphorylation of focal adhesion kinase. U251 cells expressing Ets-DN (U251-DN) showed reduced cell adhesion, spreading, and extension of actin stress fibers on dishes coated with fibronectin but not on dishes coated with collagen. Migration of U251-DN cells was found to be significantly inhibited compared with that of parental cells when examined by wound-induced migration assay on fibronectin-coated dishes. Phosphorylation levels of focal adhesion kinase in U251-DN cells were also attenuated on dishes coated with fibronectin. Reduced expression level of integrin alpha5 subunit in U251-DN cells was demonstrated by semiquantitative reverse transcription-PCR analysis. Semiquantitative reverse transcription-PCR of surgical samples of brain tumors revealed that the expression level of Ets-1 mRNA correlated with that of integrin alpha5 mRNA in glioma. The experimental metastatic ability of U251-DN cells examined in chick embryo was considerably lower than that of parental cells. These results suggest that Ets-1 contributes to glioma malignancy by up- regulating expression of the integrin alpha5 subunit, which composes integrin alpha5beta1 and mediates intracellular signaling and the subsequent acceleration of the invasive process, including cell adhesion and migration.

Calcitonin stimulation of cyclic adenosine 3':5'-monophosphate production with growth inhibition in human renal adenocarcinoma cell lines.

Responsiveness of cyclic adenosine 3':5'-monophosphate (cAMP) to parathyroid hormone, calcitonin, and vasopressin was studied in six human renal adenocarcinoma cell lines. Four of six renal adenocarcinoma cell lines showed increased cAMP content in response to calcitonin while the other two did not. Neither parathyroid hormone nor vasopressin increased the concentration of cAMP in each of these cell lines. The growth rate of KU-2 cells, which responded to calcitonin with an increase of cAMP content, was inhibited by calcitonin. On the other hand the growth rate of calcitonin-nonsensitive KH-39 cells was unaltered. The growth inhibitory effect of the hormone on KU-2 cells could be considered to be mediated by the increased cAMP levels from the following results: (a) there was positive correlation between the cellular cAMP content and growth inhibition after various amounts of calcitonin addition; (b) KU-2 growth was also suppressed by N6,O2'-dibutyryl cAMP; and (c) a group of KU-2 cells which had become resistant to calcitonin-induced growth inhibition showed a diminished cAMP increase in response to calcitonin.

Suppression of membrane-type 1 matrix metalloproteinase (MMP)-mediated MMP-2 activation and tumor invasion by testican 3 and its splicing variant gene product, N-Tes.

Using expression cloning to screen a human fetal kidney cDNA library for regulator(s) of pro-matrix metalloproteinase (MMP)-2 processing mediated by membrane-type (MT) 1 MMP, we isolated a cDNA whose product interfered with pro-MMP-2 activation. It encodes the NH(2)-terminal 313-amino acid region of a calcium-binding proteoglycan, testican 3, with a 3-amino acid substitution at the COOH terminus and thus was named N-Tes. N-Tes comprises a signal peptide, a unique domain, a follistatin-like domain, and a Ca(2+)-binding domain but lacks a COOH-terminal thyroglobulin domain and two putative glycosaminoglycan attachment sites of testican 3. Pro-MMP-2 activation by MT3-MMP was also inhibited by the coexpression of N-Tes. Immunoprecipitation analysis demonstrated direct interaction of N-Tes with either MT1-MMP or MT3-MMP. Expression of testican 1 or testican 3 but not testican 2 also inhibited pro-MMP-2 activation by either MT1-MMP or MT3-MMP. Deletion and substitution of amino acids residues in N-Tes revealed that the unique NH(2)-terminal domain of N-Tes is responsible for the inhibition of pro-MMP-2 activation by MT-MMPs. Expression of N-Tes and testican 3 was detected in normal brain but down-regulated in glioma tissues. Transfection of either the N-Tes or testican 3 gene into U251 glioma cells or Madin-Darby canine kidney cells transformed by erbB2 suppressed their invasive growth in collagen gel. These results suggest that both N-Tes and testican 3 would interfere with tumor invasion by inhibiting MT-MMPs.

Inflow Jet Patterns of Unruptured Cerebral Aneurysms Based on the Flow Velocity in the Parent Artery: Evaluation Using 4D Flow MRI.

BACKGROUND AND PURPOSE: Inflow jet characteristics may be related to aneurysmal bleb formation and rupture. We investigated the visualization threshold on the basis of the flow velocity in the parent artery to classify the inflow jet patterns observed on 4D flow MR imaging. MATERIALS AND METHODS: Fifty-seven unruptured aneurysms (24 bifurcation and 33 sidewall aneurysms) were subjected to 4D flow MR imaging to visualize inflow streamline bundles whose velocity exceeded visualization thresholds corresponding to 60%, 75%, and 90% of the maximum flow velocity in the parent artery. The shape of the streamline bundle was determined visually, and the inflow jet patterns were classified as concentrated, diffuse, neck-limited, and unvisualized. RESULTS: At the 75% threshold, bifurcation aneurysms exhibited a concentrated inflow jet pattern at the highest rate. At this threshold, the inflow jets were concentrated in 13 aneurysms (group C, 22.8%), diffuse in 18 (group D, 31.6%), neck-limited in 11 (group N, 19.3%), and unvisualized in 15 (group U, 26.3%). In 16 (28.1%) of the 57 aneurysms, the inflow jet pattern was different at various thresholds. Most inflow parameters, including the maximum inflow velocity and rate, the inflow velocity ratio, and the inflow rate ratio, were significantly higher in groups C and D than in groups N and U. CONCLUSIONS: The inflow jet pattern may depend on the threshold applied to visualize the inflow streamlines on 4D flow MR imaging. For the classification of the inflow jet patterns on 4D flow MR imaging, the 75% threshold may be optimal among the 3 thresholds corresponding to 60%, 75%, and 90% of the maximum flow velocity in the parent artery.

The plasma binding protein for vitamin D is a site of discrimination against vitamin D-2 compounds by the chick.

The binding of 25-hydroxy-[26,27-3H]vitamin D-3 and 25-hydroxy-[26,27-3H]vitamin D-2 to the vitamin D binding protein in the plasma of both rats and chicks has been studied. In the case of rats, sucrose density gradient analysis, competitive displacement, and Scatchard analysis demonstrate that 25-hydroxyvitamin D-3 and 25-hydroxyvitamin D-2 are bound equally well to the vitamin D binding protein. In contrast, 25-hydroxyvitamin D-2 is poorly bound, while 25-hydroxyvitamin D-3 is tightly bound to the vitamin D binding protein in chick plasma. On the other hand, the chick intestinal receptor binds 1,25-dihydroxyvitamin D-2 and 1,25-dihydroxyvitamin D-3 equally well with a KD of 7.10(-11) M for both compounds. These results strongly suggest that the failure of the plasma transport protein in chicks to bind the vitamin D-2 compounds may be responsible for their relative ineffectiveness in these animals.

Abciximab suppresses the rise in levels of circulating inflammatory markers after percutaneous coronary revascularization.

BACKGROUND: Previous investigators have shown that systemic markers of inflammation may be increased in patients with acute ischemic syndromes or after percutaneous coronary revascularization and that persistent elevation in these markers is predictive of excess risk of subsequent adverse cardiac events. By virtue of its cross-reactivity with the glycoprotein IIb/IIIa, avbeta3, and alphaMbeta2 receptors, abciximab may reduce inflammatory processes. Methods and Results-- Assays for the inflammatory markers C-reactive protein, interleukin-6, and tumor necrosis factor-alpha were performed on serum samples obtained from 160 patients in a placebo-controlled, randomized trial of abciximab during angioplasty. Eighty patients each had received a placebo or abciximab bolus plus a 12-hour infusion. Serum samples were drawn at baseline (before revascularization), 24 to 48 hours after study drug administration, and 4 weeks after study drug administration. Between baseline and 24 to 48 hours, the increase in C-reactive protein was 32% less in patients receiving abciximab than placebo (P=0.025); the rise in interleukin-6 levels was 76% less in the abciximab group (P<0.001); and the rise in tumor necrosis factor-alpha levels was 100% less with abciximab therapy (P=0.112). By 4 weeks, most marker levels had returned to baseline, with no significant differences between placebo and abciximab groups. CONCLUSIONS: Systemic markers of inflammation increase in the first 24 to 48 hours after angioplasty, but the magnitude of that rise is diminished by periprocedural abciximab. Some of the long-term clinical benefit derived from this agent may be related to an anti-inflammatory effect.

A chimeric IgG4 monoclonal antibody directed against CD18 reduces infarct size in a primate model of myocardial ischemia and reperfusion.

OBJECTIVES: This study attempted to determine whether neutrophil sequestration in reperfused myocardium can be inhibited and infarct size reduced by treatment with a chimeric, monoclonal IgG4 antibody (CLB54) directed against CD18 in a primate model of acute myocardial ischemia and reperfusion. BACKGROUND: Reperfusion injury, in part mediated by neutrophils, may limit the potential benefit of reestablishing infarct-related artery patency in patients with acute myocardial infarction. METHODS: Nineteen closed-chest baboons (10 control, 9 treated with CLB54) had the left anterior descending coronary artery occluded for 90 min, followed by 4 h of reflow. CLB54 (mean [+/- SD] 11 +/- 2 mg/kg body weight) or saline solution was administered intravenously 20 min before reflow. Coronary flow was determined using radiolabeled microspheres, infarct size by triphenyltetrazolium chloride staining, global and regional ventricular function by contrast ventriculography and neutrophil accumulation by a myeloperoxidase assay. RESULTS: Risk region size was the same in both groups. CLB54 treatment reduced infarct size expressed as a percent of the risk region from 41 +/- 20% in the saline-treated group to 19 +/- 17% in the CLB54-treated group (p < 0.02). This was associated with diminished myeloperoxidase activity and greater postreperfusion coronary flow in the risk region in CLB54-treated than in control baboons. Ejection fraction declined to the same extent in both groups, whereas anterior wall regional cord shortening was better preserved in CLB54-treated baboons. CONCLUSIONS: Inhibition of neutrophil sequestration with CLB54 administered before reperfusion reduces infarct size, preserves ischemic zone microvascular perfusion and minimizes the decline of regional wall motion.

Diverse coordination modes in tin analogues of a cyclopentadienyl anion depending on the substituents on the tin atom.

Reactions of an anionic heavy ruthenocene with CCl4, MeI, EtBr and Me3SiCl afforded the first stannole monoanion complexes. Surprisingly, coordination modes of the stannole rings are highly dependent on the substituents on the tin atom. The chloro derivative exhibits a η(4)-fashion-like coordination mode with a bent stannole ring, whereas the trimethylsilyl derivative adopts the conventional η(5)-coordination mode. Coordination modes of the alkyl derivatives are in between the two types. Cyclic voltammograms for these complexes reveal that the electron-donating character of the stannole ligand becomes stronger as the stannole ring becomes planar. Theoretical calculations elucidate that the different coordination modes originate from both electronegativity of an adjacent atom to the tin atom and bulkiness of a substituent on the tin atom.