Hypericin uptake: a prognostic marker for survival in high-grade glioma.

Currently adjuvant chemotherapy for glioblastoma patients can prolong survival time relative to patients who receive only surgery and radiotherapy. Despite these improvements and experimental and clinical efforts the prognosis for glioblastoma patients remains poor. At present, interest is focused on individual prognostic factors influencing patient responses to therapy. Photodynamic therapy may be a promising therapeutic option in the treatment of glioblastoma. In this investigation we examined whether uptake of hypericin (HY), a fluorescent photosensitization agent, by ex vivo glioblastoma cell lines correlates with prognosis of the individual from which the cell lines were derived. Twelve primary human glioma cell cultures were incubated with 20 micromol HY. Fluorescence intensity was measured using fluorescence microscopy. Three patients suffered from an anaplastic astrocytoma, WHO grade III, nine had a glioblastoma, WHO grade IV. In 6/12 patients complete tumour resection was possible. The mean survival time of the six patients in whom complete tumour resection was performed was 26 months, compared with 5 months for those who underwent incomplete resection. Eleven patients received radiation therapy. The five patients who received chemotherapy survived for a mean duration of 26 months, compared with the seven patients who survived for a mean duration of 5 months without chemotherapy. Statistical analysis using a parametric survival model based on the Weibull distribution showed that fluorescence intensity was the variable with the lowest p-value associated with survival (p=0.0225). An increase of 553 arbitrary units of fluorescence intensity is predicted to double survival time. Uptake of HY, a lipophilic molecule, is assumed to be related to low-density lipoprotein (LDL) uptake and metabolism. Cell proliferation is associated with a high turnover of cholesterol and membrane growth, which is related to cholesterol uptake by LDL. In summary, HY uptake by ex vivo glioblastoma cell cultures seems to be positively associated with survival of patients with malignant glioma.

Novel retractor for endoscopic and microsurgical spinal interventions.

Retractors play an important role in surgery, especially using endoscopic techniques like microendoscopic discectomy. As a consequence of the minimally invasive approach the working space is frequently very restricted. All currently available systems still use tubular retractors with limitations regarding flexibility, stability and accessible working space. This paper discusses the technical aspects of a newly designed circular retractor system for endoscopic and microsurgical spinal interventions (SpineGate) and presents first clinical results in a series of 20 consecutive patients. The autoclavable system consists of a circular base plate and several valves varying in length. The variable fixation of the valves from different directions within the 360 degrees geometry of the base plate allows a continuously adjustable working space, depending on the individual anatomy. An additional fixation device is not necessary. The retractor is suitable for microendoscopic and microsurgical techniques. It is easy and safe to handle. The retractor system was successfully used in all cases without complications or instrumental malfunctions. All patients experienced good to excellent relief of their preoperative symptoms.

Primary melanocytic lesions of the CNS: report of five cases.

Primary melanocytic tumours of the central nervous system (CNS) form a rare entity which is histologically and clinically distinct from metastatic cutaneous or retinal malignant melanoma. They can be classified into diffuse melanocytosis (diffuse melanosis), malignant melanoma and benign melanocytoma with a small number of intermediate variants. In this paper, 5 cases treated neurosurgically in our department for spinal or cerebral primary CNS malignant melanoma are reported. Primary tumors and further metastases were ruled out. Radiological, histological and clinical features are discussed. Compared to metastatic disease, primary CNS malignant melanoma shows a more benign clinical course with long-term tumour control and a good quality of life. A review of the literature which mainly consists of individual case reports, confirms this assessment. Although therapeutic experience for primary melanocytic lesions of the CNS is based on a small number of published cases, prognosis seems highly dependent on complete tumour resection. Adjuvant radiation seems to be of additional therapeutic benefit. Except for meningeosis melanomatosa chemotherapy must be regarded as experimental. Unfortunately, a standardised therapy concept is still lacking.

Anatomy of the cerebral ventricular system for endoscopic neurosurgery: a magnetic resonance study.

BACKGROUND: Endoscopy has developed into an integral part of minimally invasive neurosurgery. For further technological innovations, detailed knowledge about the pathological anatomy is essential. The gross anatomy of the cerebral ventricular system has been meticulously investigated with ventriculography and casts. Extensive volumetric measurements based on neuroradiological images have been performed, but only little is known about the surgically relevant linear distances in patients with hydrocephalus. METHOD: Thirty healthy volunteers and thirty patients suffering from hydrocephalus were scanned with high-resolution 3-D magnetic resonance imaging sequences. The image volumes were sliced identically with the help of Siemens Prominence software. Individual anatomical measurements of the ventricular system were carried out, mean values and standard deviations were calculated, and different endoscopic approaches were investigated. FINDINGS: In healthy volunteers the measurements confirmed the results obtained from ventriculography and anatomic casts. In hydrocephalic patients the ventricular system was found to be enlarged asymmetrically. The optimal neuroendoscopic approach showed considerable, interindividual variation. INTERPRETATION: This 3-D magnetic resonance imaging study revealed surgically and clinically relevant aspects of the pathologic anatomy of hydrocephalic patients, in comparison to healthy volunteers. Individualized planning of the endoscopic approach appears to be warranted. Finally, the data provided a sound basis for the further development of neuroendoscopes.

Relevance of image fusion for target point determination in functional neurosurgery.

BACKGROUND: For the treatment of medically refractory movement disorders such as Parkinson's disease, essential tremor and primary dystonia, deep brain stimulation (DBS) has become one of the main treatment options. The targets for implantation of the stimulation electrodes are various nuclei within the basal ganglia or the thalamic and subthalamic area. Accurate target localisation is of major importance for outcome and patient safety. The goal of this study was to evaluate the role of image fusion in the determination of target co-ordinates. METHOD: We conducted a retrospective study on 10 patients in whom 17 DBS electrodes had been implanted. Coordinates of the anterior and posterior commissures and of the DBS targets were compared on pre- and postoperative computerised tomography (CT) and fused CT/magnetic resonance scans. The targets as defined on the images were further compared with the targets derived intra-operatively with microelectrode recordings (MER) and macrostimulation. FINDINGS: The achievable mean target accuracy was of the order of the diameter of the DBS electrode and of the accuracy of the image fusion algorithm, i.e. about 1 mm. However, the maximal differences were between 1.8 mm and 3.2 mm. INTERPRETATION: Image fusion is a helpful tool for accurate determination of target point co-ordinates in DBS. In combination with intraoperative, electrophysiological recordings and stimulation which are still considered to be the most reliable localisation methods, image fusion may help to discern the anatomical and functional three-dimensionality of the target nuclei. Image fusion may reduce the number of trajectories needed for intraoperative electrophysiological determination of the optimal electrode localisation and thus lower the risk of complications.

Combined therapy of cerebral arteriovenous malformations: histological differences between a non-adhesive liquid embolic agent and n-butyl 2-cyanoacrylate (NBCA).

OBJECTIVE: Based on 2 casuistics, the intraoperative qualities of a new, non-adhesive liquid embolic agent (Onyx, Micro Therapeutics. Inc., Irvine, CA, USA) are to be compared to those of n-butyl 2-cyanoacrylate (NBCA) with regard to the histopathological results after preoperative embolization of a cerebral arteriovenous malformation (AVM). PATIENTS AND METHODS: In a case example, the intraoperative quality of the nidus after embolization of a parieto-occipital AVM with Onyx--a new, non-adhesive liquid embolic agent--consisting of ethylene-vinyl alcohol copolymer (EVOH), dimethyl sulfoxide (DMSO) and tantalum, is described. In the second patient, embolization of a frontal high-flow AVM was performed with NBCA. Both patients underwent surgery with complete resection ofthe AVM. RESULTS: From a neurosurgical point of view, Onyx is suitable for preoperative embolization of AVMs, because the nidus intraoperatively remains elastic and formable and can be dissected from the surrounding brain tissue quite well by microsurgical technique. Inflammatory reactions can be found mainly in the lumina of the vessels. CONCLUSIONS: Onyx promises to be an embolic agent well suitable for subsequent neurosurgical resection. Further studies considering various intervals of time between embolization and resection as well as histopathological and electron microscopical examinations are necessary for evaluation of our first experience with this new embolization agent.

Overexpression, purification, and characterization of a thermostable chitinase (Chi40) from Streptomyces thermoviolaceus OPC-520.

A new procedure for the large-scale purification of the recombinant thermostable chitinase (Chi40) cloned from Streptomyces thermoviolaceus in various expression vectors in Escherichia coli is described. Chi40 was overproduced in the cytosolic and secreted forms. The cytosolic form (Chi40c) was highly overproduced and purified by metal-affinity and ion-exchange chromatography in large amounts. The protein was highly active and thermostable but not homogeneous, since a considerable proportion of the Chi40c protein was not correctly folded as determined by native polyacrylamide gel electrophoresis. The Chi40 protein secreted into the culture medium (Chi40s) was purified by hydrophobic interaction and ion-exchange chromatography and high amounts of correctly folded and active Chi40 protein could be recovered in a short time. The enzymatic activity of Chi40s on a synthetic and on its natural substrate, chitin, was studied. Thermostability measurements showed that Chi40 has a T(m) of 60.7 degrees C at neutral pH. (13)C-(15)N double-labeled recombinant Chi40s was also produced and purified from the pECHChi40-9 construct introduced into BL21trxB(DE3) cells grown in minimal medium in the presence of the paramagnetic elements [(13)C]glucose and (15)NH(4)Cl. The presented data open the possibility of an extensive structural study on Chi40s by X-ray crystallography and on enzyme-substrate interaction by NMR spectroscopy.

New virtual system for planning of neuroendoscopic interventions.

OBJECTIVE: The demands on virtual planning systems are increasing, particularly for technically pretentious surgical interventions such as intracranial endoscopy. In this article, a new virtual system for neuroendoscopy (VIVENDI) is presented. The main purpose of this system is to provide support for planning and training in neuroendoscopic interventions. MATERIALS AND METHODS: The software is applied for virtual endoscopic visualization of three-dimensional magnetic resonance datasets, using a clinical magnetic resonance scanner. Rendering is performed on a Hewlett-Packard UNIX workstation. RESULTS: Virtual endoscopy provides a three-dimensional view of the cerebral ventricles, with good visualization of anatomic details. The rendering system used allows the generation of fly-through sequences for the entire ventricular system in real time. Navigation is controlled by mouse movements, and the visualization of the computer-generated intraventricular spaces is adapted to the characteristics of the optical endoscope. CONCLUSIONS: The presented virtual neuroendoscopy system is a promising tool for planning and training in neuroendoscopic procedures. It enables these procedures to be simulated prior to surgery based on the patient's individual anatomy.

Quality planning for minimally invasive procedures in neurosurgery.

Parallel to the introduction of a minimally invasive method in a department, a documentation system should be introduced for quality management. The first step of quality management of an innovation is quality planning. During the course of patients being treated neuroendoscopically, the pre- and postoperative imaging, the intraoperative video recording, the patient-relevant files and the data of the planning have to be documented. These amounts of data require a multimedial documentation concept. We found the CD-ROM as an optimal documentation media because the discs are both cheap and easily accessible and once stored extremely robust against external influences. Therefore, every neuroendoscopically treated patient is documented with all relevant pictures, files and video sequences on a single CD-ROM.

Early activation of the primary somatosensory cortex without conscious awareness of somatosensory stimuli in tumor patients.

The primary sensory cortex has usually been regarded as a necessary step in the information processing stream leading to conscious awareness. Recently, it has been proposed that that higher order associative areas rather than the primary sensory areas are the neural basis of conscious perception. In two patients with tumors near the central region we recorded magnetic somatosensory evoked fields. Magnetic source imaging revealed early (40 ms) neural activation in primary somatosensory cortex and absence of later (>60 ms) neural activation in the primary and associative areas in these patients. None of the patients showed conscious awareness of somatosensory stimuli applied to the corresponding body site although the first component of the evoked field was within normal limits. The time course of the magnetic responses and additional evidence on intensity ratings of somatosensory stimuli suggest that early activity in the primary somatosensory cortex is not sufficient for conscious experience to emerge.

In vivo expression of insulin-like growth factor-binding protein-2 in human gliomas increases with the tumor grade.

Human central nervous system tumors and glioma cell lines highly express the insulin-like growth factor-binding protein (IGFBP)-2. As IGFBP-2 can affect tumor growth, we studied the relationship between IGFBP-2 expression and the malignancy of brain tumors in vivo. To do so, we investigated by immunohistochemistry the accumulation of IGFBP-1, -2, and -3 in 50 human gliomas classified by the WHO Malignancy Scale. Double labeling using anti-CD68 (monocytes/macrophages), antiglial fibrillary acidic protein, and anti-CD3 (T cells) antibodies was performed to further characterize the IGFBP-1, -2, and -3(+) cells. The expression of IGFBP messenger RNAs (mRNAs) was tested by RT-PCR in tumor samples from nine gliomas of different grades and in eight cell lines representing the cellular composition of human glioma. As controls, the accumulation of IGFBP-2 was investigated in normal brain and in the rat C6 glioblastoma model. IGFBP-1 and -3 accumulated in endothelial and macrophage/microglial cells. IGFBP-2(+) macrophage/microglial and glioma cells clustered in the immediate vicinity of focal necrosis of the human gliomas as well as of the rat C6 glioblastoma. The labeling score of IGFBP-1 accumulation in endothelial cells correlated negatively (P: = 0.0229), and that of IGFBP-2 accumulation in glioma cells correlated positively (P: < 0.0006) with the tumor grade of the gliomas. In addition, RT-PCR analysis confirmed mRNA expression of IGFBP-1, -2, and -3 by the gliomas and glial cells. Small amounts of IGFBP-1 and -3 mRNA, but high amounts of IGFBP-2 mRNA, were detectable in macrophage-like and glioma cell lines. The results suggest cell type-specific accumulation of IGFBP-1, -2, and -3 in human glial tumors of the brain. The increase in IGFBP-2 expression with this malignancy suggests a role of IGFBP-2 in the biology of human gliomas.

Recurrence of a cerebral arteriovenous malformation after surgical excision.

Complete resection of a cerebral arteriovenous malformation (AVM) should eliminate the future risk of an associated intracranial bleeding. Because total removal of an AVM may be difficult to assess at the time of surgery, postoperative angiography has become the accepted standard for documenting that complete removal has been achieved. However, even angiographically confirmed excision of an AVM does not completely exclude the possibility of rebleeding. Regrowth of an AVM with subsequent haemorrhage has been documented in children and is attributed to forces acting on the immature vasculature. The authors report the case of a 21-year-old man whose AVM recurred 5 years after angiographically proven complete excision. According to the presented case, the authors emphasise that, even in adults, angiographic documentation of total removal does not always eliminate the risk of reformation of an AVM.

Cyclooxygenase (COX)-1 expressing macrophages/microglial cells and COX-2 expressing astrocytes accumulate during oligodendroglioma progression.

Cyclooxygenases (COX, prostaglandin endoperoxide synthases, PGG/H synthases) are potent mediators of edema, impeding blood flow and immunomodulation in the pathologically altered brain. Two COX iso-enzymes have been associated with brain disease, the constitutively expressed COX-1 and the cytokine-inducible COX-2. We have used single and double labeling immunohistochemistry to analyse COX-1 and COX-2 expression in twenty-six primary WHO grade II oligodendrogliomas, sixteen primary WHO grade III anaplastic oligodendrogliomas, twenty-seven matched recurrences and ten neuropathologically unaltered brains. COX-1 immunoreactivity was predominantly observed in macrophages/microglial cells. The number of COX-1 expressing macrophages/microglial cells was significantly lower in primary oligodendrogliomas than in primary anaplastic oligodendrogliomas (P<0.0001) and in anaplastic oligodendroglioma relapses (P=0.011). Patients with low COX-1 labeling scores in the primary tumors had significantly longer time to progression and overall survival (P=0.0285) than those with high COX-1 labeling scores. COX-2 immunoreactivity was predominantly observed in disseminated neurons and astrocytes. In glioblastoma multiforme relapses, accumulation of COX-2 expressing astrocytes was observed surrounding areas of focal necrosis. The number of COX-2 expressing astrocytes was significantly (P=0.0471) lower in primary oligodendrogliomas than in high grade oligodendroglioma relapses. These data provide convincing evidence for the differential accumulation of cyclooxygenase isoforms during oligodendroglioma progression in vivo.

Heme oxygenase (HO)-1 expressing macrophages/microglial cells accumulate during oligodendroglioma progression.

Heme oxygenase (HO-1, HSP32) catalyzes the oxidation of heme to biliverdin and carbon monoxide, a putative neurotransmitter. In the brain, HO-1 expression has been associated with neuroprotection during oxidative stress and hypoxia. However, consecutive downstream mediation is involved in neoangiogenesis and consequent neoplastic outgrowth. We have analyzed HO-1 expression in 69 oligodendroglioma tissue samples, in rat intracranially transplanted C6 gliomas, and neuropathologically unaltered control brains by immunohistochemistry. Double labeling experiments confirmed the nature of HO-1 expressing cells. Reverse transcription-polymerase chain reaction was used to demonstrate HO-1 gene expression. HO-1 immunoreactivity was predominantly observed in macrophages/microglial cells. The number of HO-1 expressing macrophages/microglial cells was significantly lower in primary oligodendrogliomas than in their matched relapses (P<0.0001) and lower in primary anaplastic oligodendrogliomas than in their relapses (P=0.0006). Prominent accumulation of HO-1 expressing macrophages/microglial cells was observed in perinecrotic areas of both experimental rat and human glioblastoma relapses. HO-1 expressing neurons, macrophages/microglial cells and astrocytes were scattered in areas of infiltrative tumor growth. Surprisingly, HO-1 mRNA was detected in only one glioblastoma multiforme relapse. We conclude from these data that HO-1 expressing macrophages/microglial cells accumulate during oligodendroglioma progression in areas of focal necrosis. However, overall biological function of this phenomenon remains to be determined.

A new thermoactive pullulanase from Desulfurococcus mucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme after expression in Bacillus subtilis.

The gene encoding a thermoactive pullulanase from the hyperthermophilic anaerobic archaeon Desulfurococcus mucosus (apuA) was cloned in Escherichia coli and sequenced. apuA from D. mucosus showed 45.4% pairwise amino acid identity with the pullulanase from Thermococcus aggregans and contained the four regions conserved among all amylolytic enzymes. apuA encodes a protein of 686 amino acids with a 28-residue signal peptide and has a predicted mass of 74 kDa after signal cleavage. The apuA gene was then expressed in Bacillus subtilis and secreted into the culture fluid. This is one of the first reports on the successful expression and purification of an archaeal amylopullulanase in a Bacillus strain. The purified recombinant enzyme (rapuDm) is composed of two subunits, each having an estimated molecular mass of 66 kDa. Optimal activity was measured at 85 degrees C within a broad pH range from 3.5 to 8.5, with an optimum at pH 5.0. Divalent cations have no influence on the stability or activity of the enzyme. RapuDm was stable at 80 degrees C for 4 h and exhibited a half-life of 50 min at 85 degrees C. By high-pressure liquid chromatography analysis it was observed that rapuDm hydrolyzed alpha-1,6 glycosidic linkages of pullulan, producing maltotriose, and also alpha-1,4 glycosidic linkages in starch, amylose, amylopectin, and cyclodextrins, with maltotriose and maltose as the main products. Since the thermoactive pullulanases known so far from Archaea are not active on cyclodextrins and are in fact inhibited by these cyclic oligosaccharides, the enzyme from D. mucosus should be considered an archaeal pullulanase type II with a wider substrate specificity.

Crystallization and preliminary X-ray crystallographic studies of the thermoactive pullulanase type I, hydrolyzing alpha-1,6 glycosidic linkages, from Fervidobacterium pennivorans Ven5.

Crystals of the thermoactive recombinant F. pennivorans type I pullulanase, purified from the supernatant of a Bacillus subtilis culture, have been obtained by the vapour-diffusion method in the presence of the inhibitor beta-cyclodextrin (2 mM) by mixing protein (15 mg ml(-1)) with an equal volume of crystallization solution containing 0.1 M bis-tris propane pH 6.5, 50 mM MgCl(2) and 15% polyethylene glycol 3350. Crystals diffracted to 3.0 A using conventional Cu Kalpha radiation and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 76.8, b = 96.2, c = 98. 5 A. The asymmetric unit contains one monomer. A preliminary 26% complete data set has been collected at 2.2 A resolution using synchrotron radiation.

Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasius A7: cloning and sequence analysis of five genes involved in the pathway.

Bacillus thermoglucosidasius A7 degraded phenol at 65 degrees C via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were cloned from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phenol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The larger component (50kDa) has 49% amino acid identity with the 4-hydroxyphenylacetate hydroxylase of Escherichia coli, while the smaller component (19kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary for activity. The catechol 2, 3-dioxygenase encoded by pheB has 45% amino acid identity with dmpB of Pseudomonas sp. CF600 and could be assigned to superfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. The 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, revealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydratase (2HDH), demonstrating 45% amino acid identity to the gene product of cumE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylating) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400.

Claudin-1 and claudin-5 expression and tight junction morphology are altered in blood vessels of human glioblastoma multiforme.

The aim of the study was to characterize the interendothelial junctions in tumor microvessels of five cases of human glioblastoma multiforme. In addition to morphological analysis, tumors were screened for the expression of junctional proteins, such as occludin, claudin-1, ZO-1 and catenins. The expression of the tight junction protein claudin-1 was lost in the majority of tumor microvessels, whereas claudin-5 and occludin were significantly down-regulated only in hyperplastic vessels. As shown by freeze-fracture analysis, under the conditions of tumor growth tight junction particles of endothelial cells were almost exclusively associated with the exocytoplasmic fracture face, providing evidence for a switch of the particles from the protoplasmic to the external leaflet of the endothelial membrane. These results suggest a relationship between claudin-1 suppression and the alteration of tight junction morphology, which is likely to correlate with the increase of endothelial permeability. Underlining the undifferentiated state of tumor microvessels, plakoglobin, a crucial protein for mature endothelial junctions, was not detectable in most microvessels, whereas beta-catenin was abundantly labeled. In this context, it is of particular interest that the majority of microvascular pericytes were negative for alpha-smooth muscle actin, which is a marker of differentiated pericytes, although pericytes were frequently found in electron micrographs. In conclusion, the data suggest that the increase in microvascular permeability in human gliomas, contributing to the clinically severe symptoms of brain edema, is a result of a dysregulation of junctional proteins.

Intracranial malignant B-cell lymphoma of the dura.

OBJECTIVE: Malignant B-cell lymphomas of the dura mater are very rare. A case of primary centroblastic/centrocytic lymphoma of the dura mimicking a bilateral convexity meningioma is presented. CLINICAL PRESENTATION: A 50-year-old woman was referred to our institution with a 6-month history of headache and two Jacksonian seizures. Computed tomography revealed a parafalcine and bilateral convexity lesion. Cerebral angiography and magnetic resonance imaging were performed prior to surgery. At surgery the tumor was removed subtotally. The patient was treated postoperatively by combined chemo- and radiotherapy. CONCLUSION: Laboratory studies and follow-up examinations revealed no evidence of systemic lymphoma nor of any immunocompromised state. According to the presented case combined surgery and chemoradiotherapy seems to be an effective treatment for this rare lesion.