Endovascular management of spinal dural arteriovenous fistulas in 78 patients.

INTRODUCTION: The purpose of this study was the evaluation of the safety and efficiency of the endovascular treatment of spinal dural arteriovenous fistulas (SDAVFs). METHODS: Between May 1992 and August 2012, 78 patients (59 men) with an angiographically proven SDAVF with pial venous drainage were treated by endovascular embolization (n = 61) and/or surgery (n = 31) at three German hospitals by a single team of physicians and according to a uniform therapeutic concept. RESULTS: Endovascular treatment resulted in a complete occlusion of the fistula in 47 cases (77 %). After failed embolization with residual shunt, 14 DAVFs were surgically cured (23 % failure rate). We had one permanent and two minor complications after endovascular therapy. Within a postoperative period of 2 weeks, 73.6 % of patients improved in gait disability, 51.1 % in micturition function, and 70.5 % in paresthesia of the lower extremities. Long-term follow-up data showed further improvement of clinical symptoms confirmed by normalization or resolution of spinal changes on MRI. CONCLUSIONS: An interdisciplinary approach to the management of SDAVFs is mandatory. Patients without a common origin of arteries supplying the spinal cord and the dural fistula, and without a stenosis or occlusion of the concerning segmental artery are potential candidates for endovascular treatment (diluted n-butyl-2-cyanoacrylate). Only occlusion of the "nidus" and the proximal segment of the draining vein can lead to clinical improvement.

Detection and treatment of spinal CSF leaks in idiopathic intracranial hypotension.

INTRODUCTION: This study aimed to evaluate the diagnostic imaging findings and treatment results of patients with idiopathic intracranial hypotension (IIH) due to cerebrospinal fluid (CSF) leaks. METHODS: Between February 2009 and April 2012, 26 IIH patients (15 men, median age 49 years) presenting with orthostatic headache (n = 20) and/or with spontaneous subdural effusions or subarachnoid hemorrhage (n = 19) were enrolled. Twenty-three patients underwent a whole spine CT and MRI myelography, starting 45 min after the intrathecal injection of 9 cc of iomeprol (Imeron 300 M) and 1 cc of gadobutrolum (Gadovist). Three patients only underwent MR myelography after intrathecal gadobutrolum injection. Adjacent to the level(s) of the detected CSF leak(s) along the nerve roots, 20 cc of fresh venous blood with 0.5 cc Gadovist was injected epidurally (blood patch, BP). The distribution of the BP was visualized by MRI the following day. Treatment results were evaluated clinically and by myelography 2 weeks after the application of the BP. Retreatment was offered to patients with persistent symptoms and continued CSF leakage. RESULTS: CSF leaks were detected at the cervical (n = 12), thoracic (n = 25), or lumbar (n = 21) spine. In 23 patients, more than one spinal segment was affected. One patient refused treatment. BP were applied in one (n = 9) or several (n = 16) levels. Clinical and/or radiological improvement was achieved after one (n = 16), two (n = 5), three (n = 3), or five (n = 1) BPs. CONCLUSION: CT and MRI myelography allow the reliable detection of spinal CSF leaks. The targeted and eventually repeated epidural BP procedure is a safe and efficacious treatment.

Navigated transcranial magnetic stimulation-guided resection of a left parietal tumor: case report.

BACKGROUND: Operating on tumors with close margins to the primary motor cortex requires a precise preoperative planning. Transcranial magnetic stimulation (TMS) is the only preoperative technique for detecting eloquent cortical regions that is directly comparable to direct cortical stimulation (DCS). Combining this well established method in neurological diagnostics with a non-invasive navigation system using the patient's preoperative MRI scans in the NEXTSTIM system might be a promising tool in preoperative planning. CASE REPORT: We used preoperative navigated TMS to localize the motor strip in a 75-year-old female patient with a central lesion suspected to be a meningeoma. The elicited data were fused to the navigation MRI in the Brainlab IPLAN system. Following the preoperatively acquired MEP data, a posterior approach to the tumor was planned. After craniotomy, DCS and phase reversal were performed to verify the preoperative results. The motor strip could be located in the same cortical area where the motor response was elicited preoperatively. CONCLUSION: Navigated transcranial cortical stimulation via the NEXTSTIM system is a helpful tool for preoperative planning.

Diagnostics and treatment of spontaneous intracranial hypotension.

OBJECTIVE: Intracranial hypotension is a frequently misdiagnosed syndrome which is caused by reduced intracranial cerebrospinal fluid (CSF) pressure due to spontaneous spinal CSF leakage. We present our series of intracranial hypotension regarding especially the required diagnostic imaging and the treatment. METHODS: A retrospective analysis was performed on 8 patients (5 males, 3 females, mean age 49 years) with postural and non-postural headache due to spinal CSF collection. RESULTS: Cranial MRI showed diffuse pachymeningeal gadolinium enhancement in all cases. CSF leakage detected by gadolinium-enhanced MR cisternography could be either diffuse (n=5) or precisely located around a dural tear (n=3). All but one leakages were located at the thoracic spine. In 6 patients 40-65 mL of blood were injected through epidurally placed drainages. In 1 patient, a dural tear was sealed with fibrin glue and fat. One patient refused surgical intervention. One epidural haematoma had to be revised. 5 of 7 patients showed excellent results. CONCLUSION: Gadolinium-enhanced MR cisternography best revealed CSF leaks. In the majority of patients with spontaneous intracranial hypotension, complete recovery may be achieved via a midthoracic epidural blood patch with minimal complications.

Prevention of scrapie pathogenesis by transgenic expression of anti-prion protein antibodies.

Variant Creutzfeldt-Jakob disease and bovine spongiform encephalopathy are initiated by extracerebral exposure to prions. Although prion transmission from extracerebral sites to the brain represents a potential target for prophylaxis, attempts at vaccination have been limited by the poor immunogenicity of prion proteins. To circumvent this, we expressed an anti-prion protein (anti-PrP) mu chain in Prnp(o/o) mice. Transgenic mice developed sustained anti-PrP titers, which were not suppressed by introduction of Prnp+ alleles. Transgene expression prevented pathogenesis of prions introduced by intraperitoneal injection in the spleen and brain. Expression of endogenous PrP (PrP(C)) in the spleen and brain was unaffected, suggesting that immunity was responsible for protection. This indicates the feasibility of immunological inhibition of prion disease in vivo.

The nuclear envelope prevents reinitiation of replication by regulating the binding of MCM3 to chromatin in Xenopus egg extracts.

BACKGROUND: A complex of MCM proteins is implicated in ensuring that DNA replicates only once in each cell cycle, by 'replication licensing'. The nuclear membrane is also implicated in replication licensing, but the relationship between the MCM proteins and the nuclear membrane is unclear. Here, we investigate the relationship between XMCM3 (a component of the Xenopus MCM complex), nuclear envelope permeability and the initiation of DNA replication once per cell cycle. RESULTS: Our results show that the nuclear envelope does not prevent the entry of XMCM3 into the nucleus, but that it does prevent the binding of XMCM3 to chromatin. We have also identified another component of the Xenopus MCM complex as a homologue of the Schizosaccharomyces pombe protein Cdc21. XMCM3 does not preferentially co-localize with sites of DNA replication. Instead, it is almost uniformly distributed on chromatin and is suddenly lost during replication. XMCM3 crosses intact nuclear membranes of G2-phase HeLa cells but cannot then bind to chromatin. Permeabilization of the nuclear envelope allows the binding of XMCM3 to G2-phase chromatin. We have therefore resolved replication licensing into two stages. The first requires the entry of a cytosolic 'loading factor' that is excluded by the nuclear membrane; subsequently, MCM3 can bind to chromatin in the presence or absence of a nuclear membrane, but only if the loading factor has gained access in the absence of the membrane. CONCLUSIONS: The Xenopus MCM complex contains homologues of yeast MCM2, MCM3, MCM5 and Cdc21 proteins. XMCM3 is displaced from chromatin during replication. The nuclear envelope allows entry of XMCM3 into the nucleus, but regulates its binding to chromatin; binding requires a loading factor which cannot cross the nuclear envelope. Based on these results we present a two-stage model for replication licensing.

Negative regulation of DNA replication by the retinoblastoma protein is mediated by its association with MCM7.

A yeast two-hybrid screen was employed to identify human proteins that specifically bind the amino-terminal 400 amino acids of the retinoblastoma (Rb) protein. Two independent cDNAs resulting from this screen were found to encode the carboxy-terminal 137 amino acids of MCM7, a member of a family of proteins that comprise replication licensing factor. Full-length Rb and MCM7 form protein complexes in vitro, and the amino termini of two Rb-related proteins, p107 and p130, also bind MCM7. Protein complexes between Rb and MCM7 were also detected in anti-Rb immunoprecipitates prepared from human cells. The amino-termini of Rb and p130 strongly inhibited DNA replication in an MCM7-dependent fashion in a Xenopus in vitro DNA replication assay system. These data provide the first evidence that Rb and Rb-related proteins can directly regulate DNA replication and that components of licensing factor are targets of the products of tumor suppressor genes.

Use of brain grafts to study the pathogenesis of prion diseases.

For the study of prion neurotoxicity, we used neural-grafting techniques: mice devoid of the normal host prion protein (Prnp% mice) received a neural graft and were intracerebrally infected with mouse prions. The growth and differentiation properties of neural grafts were defined. Growth of embryonic neuroectodermal tissue was optimal at gestational days 12.5-13.5. The blood-brain barrier is reconstituted after 7 weeks in most animals. Scrapie-infected PrPC-expressing grafts develop a severe spongiform encephalopathy and contain proteinase-resistant protein and infectivity. Infected grafts deliver high amounts of prions to the host brain without eliciting disease. Infected grafts show a progressive disruption of the blood-brain barrier. Following intraocular prion inoculation of a transplanted Prnp% mouse, prions do not reach the intracerebral graft, indicating that PrP expression is required for propagation along the optic tract.

A Rare Case of Isolated Cerebral Sarcoidosis Presenting as Suprasellar Mass Lesion with Salt-Wasting Hypopituitarism.

Background Sarcoidosis is a systemic disorder of unknown origin characterized by noncaseating granulomas. Clinical symptoms due to central nervous system (CNS) involvement occur in 5 to 7% of all cases; subclinical involvement is more frequent. Sole CNS involvement is very rare. Case Report A 25-year-old man presented with increasing polyuria and polydipsia over 8 weeks. Magnetic resonance imaging (MRI) revealed a supra- and infra-chiasmatic pre-thalamic mass lesion 1.0 × 1.4 × 1.4cm in diameter. Microsurgical biopsy verified a necrotizing noncaseating epithelioid cell tumor indicative for neurosarcoidosis. All symptoms dissolved within 3 months under stringent corticoid therapy. Conclusion Intracranial mass lesions as the primary and only manifestation of neuronal sarcoidosis are rare. Because conservative treatment is safe and effective, surgery is limited to biopsy and the alleviation of pressure-related symptoms to preserve neurologic function.

Transgenic and knockout mice in research on prion diseases.

Since the discovery of the prion protein (PrP) gene more than a decade ago, transgenetic investigations on the PrP gene have shaped the field of prion biology in an unprecedented way. Many questions regarding the role of PrP in susceptibility of an organism exposed to prions have been elucidated. For example mice with a targeted disruption of the PrP gene have allowed the demonstration that an organism that lacks PrPc is resistant to infection by prions. Reconstitution of these mice with mutant PrP genes allowed investigations on the structure-activity relationship of the PrP gene with regard to scrapie susceptibility. Unexpectedly, transgenic mice expressing PrP with specific amino-proximal truncations spontaneously develop a neurologic syndrome presenting with ataxia and cerebellar lesions. A distinct spontaneous neurologic phenotype was observed in mice with internal deletions in PrP. Using ectopic expression of PrP in PrP knockout mice has turned out to be a valuable approach towards the identification of host cells that are capable of replicating prions. Transgenic mice have also contributed to our understanding of the molecular basis of the species barrier for prions. Finally, the availability of PrP knockout mice and transgenic mice overexpressing PrP allows selective reconstitution experiments aimed at expressing PrP in neurografts or in specific populations of hemato- and lymphopoietic cells. Such studies have shed new light onto the mechanisms of prion spread and disease pathogenesis.

A human homologue of the yeast replication protein Cdc21. Interactions with other Mcm proteins.

We present the amino acid sequence of the human homologue of the yeast replication protein Cdc21, a member of the Mcm family of nuclear proteins. Specific antibodies, raised against protein hCdc21, were used to investigate the expression of the protein through the cell cycle. The protein is highly phosphorylated in mitotic cells. The phosphorylated form of protein hCdc21 appears to be less tightly bound to nuclear structures than the underphosphorylated form suggesting that phosphorylation/dephosphorylation reactions may determine the nuclear distribution of the protein. Protein hCdc21 forms a stable trimeric complex with two novel human Mcm proteins, p85Mcm and p105Mcm. Protein BM28/Mcm2 is more loosely associated with the trimeric hCdc21 complex.

Stability of the replicative Mcm3 protein in proliferating and differentiating human cells.

Mcm proteins are abundant nuclear proteins involved in the regulation of genome replication. Previous experiments had shown that levels of Mcm-specific mRNAs increase at the G1/S phase transition of the cell cycle, but that the amounts of Mcm proteins do not change much during the cell cycle. To learn more about the stability of an Mcm protein we performed experiments which showed that: (i) more than 60% of [35S]methionine pulse-labeled Mcm3 protein appears to be degraded during a 24-h chase in HeLa cells; (ii) the amount of Mcm3 protein significantly decreases during the differentiation of HL60 cells in vitro (whereas another replication-initiation protein, hOrc2, remains fairly constant); and (iii) according to immunohistochemical staining, Mcm3 protein is present in nuclei of cells in the proliferating zone of human epidermal tissue, but in decreasing amounts in nuclei of differentiating cells of the upper cell layers. Our interpretation is that Mcm3 protein is no longer synthesized after initiation of differentiation and slowly disappears at a half-life of approximately 24 h.

Human replication proteins hCdc21, hCdc46 and P1Mcm3 bind chromatin uniformly before S-phase and are displaced locally during DNA replication.

Members of the Mcm-protein family have recently been shown to be involved in restricting DNA replication to a single cycle in Xenopus laevis egg extracts. In this study, we extended these observations to human somatic cells and analysed the localisation of the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 in replicating HeLa cell nuclei. These Mcm-proteins are entirely nuclear in interphase cells and apparently exist in two populations: a nucleosolic population, and a population bound to a nuclear structure, most likely chromatin. The bound population is detected throughout the nucleus in late G1 and early S, and at discrete subnuclear sites following further progression of S-phase. We use high resolution confocal microscopy to determine the subnuclear sites of chromatin-bound Mcm proteins in comparison to the sites of replicating DNA. Importantly, hCdc21, hCdc46 and P1Mcm3 do not colocalise with replication foci, instead these proteins appear to coincide with subnuclear sites of unreplicated chromatin. During progression of S-phase hCdc21, hCdc46 and P1Mcm3 are displaced from their site on chromatin at the time when this site is replicated. Consequently, early replicating sites do not contain bound hCdc21, hCdc46 or P1Mcm3 during later stages of S-phase. Furthermore, G2 nuclei and condensed chromatin in mitotic cells do not contain bound hCdc21, hCdc46 or P1Mcm3. Thus, the human Mcm-proteins Cdc21, Cdc46 and P1Mcm3 are not concentrated at sites of DNA replication. Instead, they appear to be present only on unreplicated chromatin and are displaced from replicating chromatin, consistent with a role in monitoring unreplicated chromatin and ensuring only a single round of DNA replication per cell cycle.

Expression, phosphorylation and nuclear localization of the human P1 protein, a homologue of the yeast Mcm 3 replication protein.

The human protein P1 belongs to a newly discovered class of mammalian nuclear proteins with high sequence homology to yeast replication proteins. We present the entire amino acid sequence of the human protein P1 as predicted from the cDNA sequence, and show that P1 shares three central regions of high sequence similarity (about 75%) and a highly hydrophilic carboxy-terminal region with the yeast Mcm3 replication protein. The human genome most probably contains one P1 gene which is activated when HeLa cells progress to S phase, as shown by a several-fold increase in P1-specific mRNA. However, the amounts of P1 protein do not detectably change during this period, but P1 protein becomes phosphorylated at the beginning of S phase. In contrast to the yeast Mcm proteins, which disappear from nuclei after initiation of DNA replication, protein P1 remains in the nucleus during and after S phase. P1 is dispersed in mitotic cells and may be excluded from binding to chromosomes.

Interactions of human nuclear proteins P1Mcm3 and P1Cdc46.

Human nuclear proteins P1Mcm3 and P1Cdc46 have high sequence similarities with the corresponding yeast proteins known to be required for the initiation of genome replication. Nuclei of proliferating HeLa cells contain relatively high amounts of P1Mcm3 (about 10(6) molecules/nucleus) of which only a small fraction is bound to a nuclear structure, most probably chromatin. At 0.5 M NaCl, the structure-bound nuclear protein can be partially solubilized as a dimer composed of P1Mcm3 and the related protein P1Cdc46. However, most protein P1Mcm3 is not bound to a nuclear structure and appears in the nucleoplasm. About 10% of protein P1Mcm3 in the soluble fraction is free and uncomplexed, and the remaining P1Mcm3 forms stable complexes with protein P1Cdc46. These P1Mcm3/Cdc46 complexes occur as dimers and in high-molecular-mass complexes (approximately 500 kDa). The high-molecular-mass complexes dissociate in 0.5 M NaCl and release P1Mcm3/Cdc46 dimers. It has frequently been proposed that the Mcm proteins may function as licensing factors for genome replication. Our data imply that the active form of an Mcm protein is not a monomer, but a protein complex that includes an Mcm3/Cdc46 dimer. DNA polymerase alpha is not a component of this complex.

Coding sequence and chromosome mapping of the human gene (CDC46) for replication protein hCdc46/Mcm5.

We have isolated the complete cDNA sequence encoding the human homolog of the yeast replication protein Cdc46/Mcm5. The cDNA was used as a probe for chromosomal fluorescence in situ hybridization (FISH), which localized the human Cdc46 gene (CDC46) to chromosome region 22q13.1-->q13.2. This is the fourth human Mcm gene whose chromosome region has been determined, and it is now apparent that human Mcm genes are widely distributed in the genome. As deduced from the cDNA sequence, human protein hCdc46 is composed of 734 amino acids and contains a central region that is almost 80% identical with the yeast protein. Immunoprecipitation experiments with hCdc46-specific antibodies confirm that essentially all nuclear hCdc46 proteins form a stable dimeric complex with protein P1Mcm3.

Properties of the human nuclear protein p85Mcm. Expression, nuclear localization and interaction with other Mcm proteins.

Recently we identified a cDNA fragment encoding a conserved part of a new human minichromosome maintenance (Mcm) protein, provisionally termed P1.1Mcm3. Here, we report that the protein is most highly related to a yeast cell-division-cycle protein, Cdc47, encoded by the open reading frame YBR1441 on chromosome 11 of Saccharomyces cerevisiae. The human protein migrates on a polyacrylamide gel with an apparent molecular mass of 85 kDa and shares areas of significant similarity with the Mcm family of replication proteins. It is, therefore, designated as p85Mcm. Microscopic immuno-fluorescence studies revealed that protein p85Mcm is located in the nuclei of interphase cells, but is evenly distributed throughout the cell during mitosis. The amounts of p85Mcm do not significantly change during the cell cycle, but mRNA levels rise with the beginning of the S phase. However, in vitro differentiation of HL60 cells results in a striking decrease of both p85Mcm mRNA and protein levels, suggesting a role for p85Mcm in proliferating, but not in differentiated cells. Under physiological salt conditions, p85Mcm is a component of a high molecular-mass complex including other Mcm proteins. The complex dissociates at high ionic strength giving rise to stable subcomplexes, one of which contains protein p85Mcm together with Mcm proteins hCdc21 and p1O5Mcm.

Human minichromosome maintenance proteins and human origin recognition complex 2 protein on chromatin.

Minichromosome maintenance (Mcm) proteins and the constituents of the origin recognition complex (Orc) are essential components of the eukaryotic replication initiation apparatus. Published evidence strongly suggests that the binding of Mcm proteins to chromatin is contingent upon the prior binding of Orc proteins. Here we use two different approaches to investigate the presence of the human ORC2 protein and of Mcm proteins on chromatin of HeLa cells in various cell cycle phases. First, we mobilized chromatin-bound proteins by micrococcal nuclease and analyzed the resulting digestion products by sucrose gradient centrifugations. Under digestion conditions when Mcm proteins were almost entirely released from chromatin, ORC2 protein was found to be associated with chromatin fragments containing several hundred base pairs of DNA. Second, we used an in vivo cross-linking procedure to covalently link Mcm proteins and ORC2 to DNA by short exposure of intact HeLa cells to formaldehyde. Specific immunoprecipitations revealed that cross-linked nucleoprotein fragments carried either Mcm proteins or ORC2 protein, but not both. Based on the lengths of the DNA fragments in immunoprecipitates, we estimate that the distance between chromatin-bound ORC2 protein and chromatin-bound Mcm proteins must be at least 500-1000 base pairs in HeLa cells.

The P1 family: a new class of nuclear mammalian proteins related to the yeast Mcm replication proteins.

Monospecific antibodies against an oligopeptide, conserved among the Mcm class of yeast replication proteins, were used to screen a human cDNA library. Eight of the isolated cDNA clones have the potential to code for sections of proteins with high sequence similarities to the yeast proteins Mcm3 and Cdc46 from Saccharomyces cerevisiae and Cdc21 from S. pombe. Our results establish a novel and highly conserved family of nuclear proteins in mammalian cells.

Immunohistochemical detection of cell growth fraction in formalin-fixed and paraffin-embedded murine tissue.

Monoclonal antibody MIB-1 is a reliable tool for determining proliferating cells in human tissues, but does not react with the homologous mouse antigen and is therefore useless in experimental pathology using mice as model systems. Standard method for assessment of cellular proliferation in formalin-fixed, paraffin-embedded murine tissues is immunohistochemical detection of DNA synthesis using antibodies against exogenously injected 5-bromodeoxyuridine (BrdU), which is a tedious procedure and not useful for routine investigations. We tested monoclonal antibody MIB-5 and monoclonal and polyclonal anti-MCM3 antibodies as immunohistochemical proliferation markers for paraffin-embedded nonneoplastic and neoplastic tissues of wild-type and transgenic mice, compared to anti-BrdU immunostaining. Percentage of proliferating cells was determined with continuously decreasing antibody dilutions. Percentages of MIB-5 and anti-BrdU immunostained cells correlated strongly, as well as percentage of MIB-5-decorated cells and frequency of mitotic figures. Anti-MCM3 antibodies labeled significantly higher percentages of cells than anti-BrdU or MIB-5, and showed a linear decrease with increasing antibody dilutions. We conclude that MIB-5 detects reliably the cell growth fraction in formalin fixed, paraffin-embedded murine tissues, bypassing methodological drawbacks of BrdU. Anti-MCM3 antibodies are less useful for determination of proliferating cells although they might detect the fraction of cells remaining competent for proliferation.