Methiothepin downregulates SNAP-23 and inhibits degranulation of rat basophilic leukemia cells and mouse bone marrow-derived mast cells.

In the present study, we found that methiothepin (a nonselective 5-hydroxytryptamine [5-HT] receptor antagonist) inhibited antigen-induced degranulation in rat basophilic leukemia cells and mouse bone marrow-derived mast cells. Although antigen stimulation induces release of histamine and serotonin (5-HT) by exocytosis and mast cells express several types of 5-HT receptor, the detailed role of these receptors remains unclear. Here, pretreatment of cells with methiothepin attenuated increased intracellular Ca2+ concentration, phosphorylated critical upstream signaling components (Src family tyrosine kinases, Syk, and PLCγ1), and suppressed TNF-α secretion via inhibition of Akt (a Ser/Thr kinase activated by PI3K)and ERK phosphorylation. Furthermore, it inhibited PMA/ionomycin-induced degranulation; this finding suggested that methiothepin affected downstream signaling. IκB kinase ß phosphorylates synaptosomal associated protein 23, which regulates the fusion events of the secretory granule/plasma membrane after mast cell activation, resulting in degranulation. We showed that methiothepin blocked PMA/ionomycin-induced phosphorylation of synaptosomal associated protein 23 by inhibiting its interaction with IκB kinase ß. Together with the results of selective 5-HT antagonists, it is suggested that methiothepin inhibits mast cell degranulation by downregulating upstream signaling pathways and exocytotic fusion machinery through mainly 5-HT1A receptor. Our findings provide that 5-HT antagonists may be used to relieve allergic reactions.

A Simple Method for Purified Primary Culture of Enteric Glial Cells from Mouse Small Intestine.

Enteric glial cells (EGCs) have been recognized as an important cell type constituting the enteric nervous system. EGCs control intestinal function and homeostasis through interactions with enteric neurons, epithelial cells and immune cells. To clarify the roles of EGCs in intestinal function and homeostasis, especially through secretion of and response to physiologically active substances, purified EGCs in primary culture have great advantages as an experimental tool. However, contamination by other cell types, fibroblasts in particular, is problematic in conventional primary myenteric culture. Previous methods to purify primary EGCs take a long time (over one month), are expensive, and are labor intensive. In the present study, we sought to purify primary EGCs from mouse small intestine by a simpler method than previous ones. After trying various protocols, we have established a method combining serum-free treatment and scraping fibroblasts off with a pipette tip. With our method, a purity of more than 90% EGCs was achieved after 14-d primary culture. Thus, our method is useful for investigating the roles of EGCs in intestinal function and homeostasis in detail in vitro.

Encapsulation of Plant Viral Particles in Calcite Crystals.

The concept of biomineralization and encapsulation of organic molecules into inorganic matrices to alter and enhance their physical properties has been evolved and perfected in natural systems. Being inspired by the natural biomineralization of foreign components into calcite, here the inclusion of a plant virus, cowpea mosaic virus (CPMV) of 5.4% by mass into crystals of calcite is reported. The viral particles are labeled with a fluorescent tag (Alexa Fluor 532), and are observed within the calcite matrix using confocal fluorescence microscopy. Upon encapsulation, the calcite crystals exhibit an irregular and aggregated morphology, as visualized with atomic force and electron microscopy. The viral particles protected inside the calcite crystals are able to resist harsh chemical agents. While spherical viral particles such as CPMV can be easily included in calcite, viruses such as the tobacco mosaic virus are not compatible with the host, presumably due to their high aspect ratio. The results provide a simple and scalable method to incorporate viral particles into inorganic matrix, and could prove useful in thermal stabilization of sensitive viral biological agents such as vaccines in the future.

Stabilizers of edaravone aqueous solution and their action mechanisms. 2. Glutathione.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has garnered attention since its approval for amyotrophic lateral sclerosis in Japan (2015) and the United States (2017). Edaravone is administered intravenously, and as such, is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because they present as edaravone anions, which become edaravone radicals when the anion donates an electron to free radicals including oxygen. In this study, glutathione (GSH) stabilized an aqueous edaravone solution during storage at 60°C for 4 weeks, and prevented the formation of potentially carcinogenic phenylhydrazine, while cysteine did not. One possible explanation is that GSH undergoes intermolecular hydrogen bonding with edaravone anions, while cysteine does not, as it favors intramolecular hydrogen boding. The combination of GSH and sodium bisulfite (NaHSO3) stabilized aqueous edaravone at room temperature for more than 1 year even under aerobic conditions. However, the U.S. Food and Drug Administration cautioned that NaHSO3 may cause allergic reactions. Therefore, we developed a stable edaravone aqueous solution without using NaHSO3, namely a combination of GSH with deoxygenation, which resulted in better stabilization of aqueous edaravone than the combination of GSH and NaHSO3.

Successful Hybrid Neurovascular Surgery for a Ruptured Basilar Artery Dissecting Aneurysm in a Patient with Jeopardized Collateral Circulation Associated with Aortitis Syndrome.

BACKGROUND: Dissecting aneurysms of the basilar artery (BA) are rare lesions, and the management of them has been controversial and challenging. The treatment becomes much more difficult when the patient has a disorder such as aortitis syndrome, which makes cerebral arteries occluded and tortuous. We describe a case of a ruptured BA dissecting aneurysm associated with aortitis syndrome, successfully treated with hybrid neurovascular surgery. CASE DESCRIPTION: A 64-year-old woman with a medical history of aortitis syndrome developed a severe headache and was diagnosed with a subarachnoid hemorrhage from a BA dissecting aneurysm. Her right common carotid artery and right vertebral artery (VA) were occluded because of aortitis syndrome. Blood flow in the right middle cerebral artery (MCA) was mainly supplied by the right posterior communicating artery. It was essential to preserve blood flow in the BA to prevent a cerebral infarction in the area of the right MCA. Stent-assisted coil embolization was considered the most appropriate treatment. The occluded right VA was thought to be more appropriate for introduction of an intravascular stent than the left VA. The patient underwent open surgery to expose the right VA. An intravascular stent was placed through the exposed right VA, and intra-aneurysmal coils were introduced from the left VA. The aneurysm was successfully treated with a hybrid technique. CONCLUSIONS: To our knowledge, this is the first report of hybrid neurovascular surgery for a BA dissecting aneurysm. A ruptured BA dissecting aneurysm in a patient with aortitis syndrome was successfully treated with hybrid neurovascular surgery.

A case of cervicogenic headache caused by C5 nerve root derived shwannoma: Case report.

Introduction We report a case of cervicogenic headache caused by an intradural extramedullary tumor of the middle cervical spine, which has not previously been reported. Case presentation The patient was a 73-year-old male who visited a physician for a chief complaint of pain from the left lower jaw to the auricle and occipital region. The headache was induced with retroflexion of the neck. On cervical magnetic resonance imaging, an intradural extramedullary tumor was noted on the left side at the C4/5 level. The intradural tumor, which arose from the C5 nerve root, was excised and the pain was resolved. The pathological diagnosis was schwannoma. Conclusion Previously reported cases of spinal cord tumor-induced cervicogenic headache were due to upper cervical spinal tumors. This is the first report that a middle-lower cervical intradural extramedullary tumor caused cervicogenic headache.

[Pediatric medulloblastoma presenting as cerebellar hemorrhage: a case report].

Medulloblastomas usually cause cerebellar ataxia and acute hydrocephalus owing to their increase in size. Cerebellar hemorrhage is an extremely rare initial clinical presentation of medulloblastoma. Herein, we report a case of medulloblastoma in an 8-year-old girl who presented with initial cerebellar intratumoral hemorrhage. The patient initially presented with mild headache;the differential diagnosis by using the initial computed tomography and magnetic resonance images was difficult, as bleeding from a cerebellar vascular malformation(cavernous angioma or arteriovenous malformation)was considered more likely. Hydrocephalus or typical findings indicative of medulloblastoma were not observed. We initially only observed the patient at another institution because the hematoma was relatively small(1.5×1×1cm). After follow-up imaging for pathological diagnosis, surgical removal was performed at our institute 49 days after the hemorrhage was observed. Complete tumor removal was achieved, and the histopathological diagnosis was medulloblastoma. The patient received whole brain and spinal irradiation(23.4Gy;posterior fossa local:50.4Gy)and chemotherapy(cyclophosphamide, 1,000mg/m2/day on day 1;vincristine, 1.5mg/m2/day on day 1;etoposide, 100mg/m2/day on days 1-3;cisplatin, 90mg/m2/day on day 2). No recurrences or neurological deficits were observed during a 2-year follow-up. This was a rare case of medulloblastoma presenting as cerebellar hemorrhage. Cerebellar medulloblastoma is among the common pediatric brain tumors;therefore, it should be diagnosed accurately and quickly.

Rosette-forming glioneuronal tumor of the fourth ventricle with neurocytoma component.

Rosette-forming glioneuronal tumor (RGNT) was first published in 2002 and was described as a benign and indolent tumor. It was also included in the 2007 World Health Organization (WHO) classification of tumors as a grade 1 tumor for its benign clinical behavior and the possibility of surgical cure. Pathologically, RGNT is a mixed neuronal-glial tumor which consists of two distinct histological components-one with uniform neurocytes forming rosettes and/or perivascular pseudorosettes and the other being astrocytic in nature resembling pilocytic astrocytoma (biphasic pattern). We present the clinical course and pathological findings of two distinctively different cases. The first one was a 4-year-old girl with head trauma and a tumor which was incidentally found by CT. Pathology revealed that the tumor contained neurocytoma components and areas of relatively high proliferative ability with the first report of the presence of midsized bright elliptic cells. The other case was a 19-year-old girl whose imaging studies showed hydrocephalus and a brain stem tumor. She underwent endoscopic third ventriculostomy and biopsy, followed by observation. An MRI taken 6 months later showed progression of the tumor and she subsequently had the tumor excised. We are considering the possibility for our RGNT cases to correspond to a higher WHO grade as they have shown rapid progression, contrary to the already established, and their character, origin, differential diagnosis, and treatment plans have been discussed.

Diagnostic and management difficulties in a case of multiple intracranial juvenile xanthogranuloma.

INTRODUCTION: Juvenile xanthogranuloma (JXG) preferentially occurs in childhood, and its characteristics have been thought to be benign and with slow growth. JXG is classified as an inflammatory disease, which forms multiple lesions in the patients' head, neck, and other organs and typically skin. JXG is rare, and few case reports have been published in the past, particularly with multiple intracranial lesions, which as in our case, is an extremely rare finding. CASE: Patient is a 4-year-old boy who presented with polydypsia and polyuria for 1 year. He had been followed up only under l-deamino-8-D-arginine vasopressin until he started vomiting and his level of consciousness deteriorated. Then, magnetic resonance imaging (MRI) revealed multiple intracranial lesions. He underwent biopsy via small craniotomy, and pathology was confirmed as juvenile xanthogranuloma. The patient received chemotherapy, in complete compliance with JLSG-02 protocol. FINDINGS: JXG is characteristic with homogenous enhancement with contrast matter and, most importantly, high intensity on diffusion-weighted image (DWI). The DWI was high when the degree of diffusion of water is restricted, as seen in inflammation and, additionally, the change of intensity after administration of steroid would reflect its anti-inflammatory nature. However, though the steroid therapy made high-intensity lesions decrease signal intensity, the size and the number of lesions did not change at all. As we expected, after we stopped steroid administration to the patient, his consciousness deteriorated and we found again the very-high-intensity lesions. We consider the intensity on DWI to reflect progression of the lesions.

Clinicopathological features of sellar region xanthogranuloma: correlation with Rathke's cleft cyst.

Xanthogranuloma of the sellar region is a rare clinical observation. Although it was included in the World Health Organization (WHO) brain tumor classification in 2000, its clinical features and pathogenesis remain uncertain. We report herein seven cases of xanthogranuloma of the sellar region who underwent transsphenoidal surgery at Tokyo Women's Medical University between 2005 and 2011, and discuss the clinical characteristics of this tumor. Six out of these 7 patients (86 %) presented with endocrinological dysfunction, six (86 %) had headaches, five (71 %) had visual field disturbances, and three (43 %) had diplopia including two with unilateral ptosis. Pathological findings revealed cholesterol clefts, hemosiderin deposits, chronic inflammatory infiltrates, multinucleated giant cells, macrophages, and fibrous proliferation. Of these seven cases, components of Rathke's cleft cyst were observed for six. Xanthogranuloma in the sellar region is suspected of being a terminal stage resulting from a secondary reaction caused by repeated inflammatory change, hemorrhage, and degeneration of a Rathke's cleft cyst.

Effects of alfacalcidol on back extensor strength gained through back extensor exercise in postmenopausal women with osteoporosis.

OBJECTIVE: The aim of this study was to investigate the additive effect of the active form of vitamin D3 on the gain in back extensor strength through a back extensor exercise. DESIGN: A total of 107 postmenopausal women with osteoporosis were randomly divided into two groups: the D3 group and the control group. Both groups were treated with calcium and alendronate and undertook the back extensor exercise. Alfacalcidol was prescribed only to the D3 group. RESULTS: There was no significant difference in the demographic data between the two groups. Ninety-four participants who completed a 4-mo intervention were subjected to per-protocol analysis. There was no significant difference in the improvement in back extensor strength between two the groups (P = 0.349). All subjects were further categorized into two subgroups by age. In the older subgroup (≥68 yrs), no significant difference was found in the improvement in back extensor strength (P = 0.316). In the younger subgroup (<68 yrs), the back extensor strength in the D3 group was significantly more improved than in the control group (P = 0.034). CONCLUSIONS: The results of this study suggest that the administration of the active form of vitamin D3 enhances the beneficial effects of the back extensor exercise in patients younger than those in their late 60s.

Detection of asymmetric distribution of phospholipids by fluorescence resonance energy transfer.

It is well established that the plasma membrane exhibits an asymmetric distribution of lipids between the inner and outer leaflets of the lipid bilayer. Recent studies suggest that the asymmetric distribution changes locally and temporarily, accompanied by cellular events. However, available methods to detect lipid asymmetry lack spatio-temporal resolution. As a technique of potential use for real-time imaging of lipid asymmetry, we a novel method that utilizes fluorescence resonance energy transfer (FRET) between NBD-labeled phospholipids (donor) and extracellular rhodamine (acceptor). When cell apoptosis was induced by staurosporine, the fluorescence intensity of NBD-labeled phosphatidylserine decreased owing to FRET from NBD to rhodamine. This method provides a simple way to detect lipid asymmetry and may be useful for observing dynamic changes in asymmetric distribution of lipids.

Wireless modification of the intraoperative examination monitor for awake surgery.

The dedicated intraoperative examination monitor for awake surgery (IEMAS) was originally developed by us to facilitate the process of brain mapping during awake craniotomy and successfully used in 186 neurosurgical procedures. This information-sharing device provides the opportunity for all members of the surgical team to visualize a wide spectrum of the integrated intraoperative information related to the condition of the patient, nuances of the surgical procedure, and details of the cortical mapping, practically without interruption of the surgical manipulations. The wide set of both anatomical and functional parameters, such as view of the patient's mimic and face movements while answering the specific questions, type of the examination test, position of the surgical instruments, parameters of the bispectral index monitor, and general view of the surgical field through the operating microscope, is presented compactly in one screen with several displays. However, the initially designed IEMAS system was occasionally affected by interruption or detachment of the connecting cables, which sometimes interfered with its effective clinical use. Therefore, a new modification of the device was developed. The specific feature is installation of wireless information transmitting technology using audio-visual transmitters and receivers for transfer of images and verbal information. The modified IEMAS system is very convenient to use in the narrow space of the operating room.

Phase I/IIa trial of autologous formalin-fixed tumor vaccine concomitant with fractionated radiotherapy for newly diagnosed glioblastoma. Clinical article.

OBJECT: The objective of the present study was analysis of results of the prospective clinical trial directed toward the evaluation of therapeutic efficacy of the administration of autologous formalin-fixed tumor vaccine (AFTV) concomitant with fractionated radiotherapy in cases of newly diagnosed glioblastoma multiforme. METHODS: Twenty-four patients were enrolled into the clinical trial, while 2 cases were excluded from the final analysis of results. The treatment protocol included aggressive tumor resection, fractionated radiotherapy up to a total dose of 60 Gy, and 3 concomitant courses of AFTV administered with an interval of one week at the late stage of irradiation. Two delayed-type hypersensitivity (DTH) tests were done--one 48 hours before the initial course of vaccination (DTH-1) and one 2 weeks after the third (DTH-2). All but one of the patients received salvage therapy at the time of tumor progression. The defined primary end point was overall survival; secondary end points were progression-free survival and safety of concomitant treatment. RESULTS: The median duration of overall survival was 19.8 [corrected] months (95% CI 13.8-31.3 months). The actuarial 2-year survival rate was 40%. The median duration of progression-free survival was 7.6 months (95% CI 4.3-13.6 months). Overall survival showed a statistically significant association with recursive partitioning analysis class (p < 0.05); progression-free survival showed a statistically significant association with p53 staining index (p < 0.05) and size of DTH-2 response (p < 0.001). AFTV injection concomitant with fractionated radiotherapy was well tolerated by all patients and in no case did treatment-related adverse effects exceed Grade 1 toxicity; adverse effects were limited to local erythema, induration, and swelling at the site of injection. CONCLUSIONS: The results of this study demonstrate that AFTV treatment concomitant with fractionated radiotherapy may be effective in patients with newly diagnosed glioblastoma. Further clinical testing is warranted.

Information-guided surgical management of gliomas using low-field-strength intraoperative MRI.

BACKGROUND: Contemporary technological developments revolutionized management of brain tumors. The experience with information-guided surgery of gliomas, based on the integration of the various intraoperative anatomical, functional, and histological data, is reported. METHODS: From 2000 to 2009, 574 surgeries for intracranial gliomas were performed in our clinic with the use of intraoperative MRI (ioMRI) with magnetic field strength of 0.3T, updated neuronavigation, neurochemical navigation with 5-aminolevulinic acid, serial intraoperative histopathological investigations of the resected tissue, and comprehensive neurophysiological monitoring. Nearly half of patients (263 cases; 45.8%) were followed more than 2 years after surgery. FINDINGS: Maximal possible tumor resection, defined as radiologically complete tumor removal or subtotal removal leaving the residual neoplasm within the vital functionally-important brain areas, was attained in 569 cases (99.1%). The median resection rate constituted 95%, 95%, and 98%, for WHO grade II, III, and IV gliomas, respectively. Actuarial 5-year survival was significantly worse in WHO grade IV gliomas (19%), but did not differ significantly between WHO grade III and II tumors (69% vs. 87%). CONCLUSIONS: Information-guided management of gliomas using low-field-strength ioMRI provides a good opportunity for maximal possible tumor resection, and may result in survival advantage, particularly in patients with WHO grade III neoplasms.

Expression of hydroxyindole-O-methyltransferase enzyme in the human central nervous system and in pineal parenchymal cell tumors.

Pineal parenchymal tumor (PPT) cells usually show immunoreactivity for synaptophysin, neuron-specific enolase, neurofilament protein, class III beta-tubulin, tau protein, PGP9.5, chromogranin, serotonin, retinal S-antigen, and rhodopsin, but these markers are not specific for PPTs. Melatonin is produced and secreted mainly bypineal parenchymal cells; hydroxyindole-O-methyltransferase (HIOMT) catalyzes the final reaction in melatonin biosynthesis. We hypothesized that HIOMT could serve as a tumor marker of PPTs, and we investigated HIOMT localization and HIOMT expression in samples of normal human tissue and in PPTs, primitive neuroectodermal tumors, and medulloblastomas. In normal tissue, HIOMT was expressed in retinal cells, pineal parenchymal cells, neurons of the Edinger-Westphal nucleus, microglia, macrophages, thyroid follicular epithelium, principal and oxyphil cells of parathyroid gland, adrenal cortical cells, hepatic parenchymal cells, renal tubule epithelium, and enteroendocrine cells of stomach and duodenum. The HIOMT was also expressed in all 46 PPTs studied. The proportions of HIOMT-immunoreactive cells successively decreased in the following tumors: pineocytoma, pineal parenchymal tumor of intermediate differentiation, and pineoblastoma. A few HIOMT-immunoreactive cells were observed in one of 6 primitive neuroectodermal tumors and 23 of 42 medulloblastomas. These results indicate that HIOMT immunohistochemistry may be useful for the diagnosis of PPTs and be a prognostic factor in PPTs.

Sterile alpha motif containing domain 9 is involved in death signaling of malignant glioma treated with inactivated Sendai virus particle (HVJ-E) or type I interferon.

Malignant glioma is one of the most aggressive cancers. For the development of effective therapeutic strategies against such malignant diseases, elucidation of molecular targets is necessary. We found that inactivated Sendai virus particle (HVJ-E) induced extensive cell death in the human glioblastoma cell line U251MG. Intradermal U251MG tumors were more effectively suppressed by HVJ-E than interferon (IFN)-beta. From microarray analysis of gene expression in U251MG cells treated with HVJ-E, we focused on the up-regulation of sterile alpha motif containing domain 9 (SAMD9) gene. The expression of the SAMD9 gene was induced by administration of recombinant human IFN-alpha, -beta or -gamma. The up-regulation of the SAMD9 gene by HVJ-E treatment was abrogated by IFN receptor blocking antibody or JAK inhibitor treatment. When SAMD9 expression was knocked down by RNA interference, apoptotic cell death induced by HVJ-E was blocked in U251MG cells. Suppression of SAMD9 using SAMD9 siRNA also inhibited IFN-beta-induced death in U251MG cells with a small, but significant, difference to control groups. However, overexpression of the SAMD9 gene failed to induce significant cell death in U251MG cells. Thus, SAMD9 could be a key molecule to control cancer cell death by HVJ-E or IFN-beta treatment.

Role of proton magnetic resonance spectroscopy in preoperative evaluation of patients with mesial temporal lobe epilepsy.

A retrospective study was conducted for evaluation of the role of single-voxel proton magnetic resonance spectroscopy (1H-MRS) in preoperative investigation of patients with mesial temporal lobe epilepsy (MTLE). Eighteen cases, including both non-lesional and lesional MTLE, were analyzed. Selective amygdalohippocampectomy was performed in 8 cases, selective amygdalohippocampectomy combined with lesionectomy in 6 cases, lesionectomy in 3 cases, and anterior temporal lobe resection in one case. The length of follow-up varied from 24 to 71 months (median, 35 months). Before surgery, (1)H-MRS disclosed decrease of N-acetylaspartate (NAA) content (P=0.01) and more frequent (P=0.07) presence of lactate (Lac) on the side of the epileptogenic zone. Decrease of NAA content below 0.75 and/or unilateral presence of Lac provided 86% (95% CI: 68%-100%) lateralization accuracy. Metabolic parameters did not differ in subgroups with hippocampal sclerosis and brain tumors. On the long-term follow-up 12 patients (67%) were free of disabling seizures. There was a trend (P=0.05) for worse seizure outcome in cases with significant bilateral metabolic alterations characterized by predominance of choline-containing compounds' peak on 1H-MR spectra on both sides. In conclusion, 1H-MRS-detected reduction of NAA content and unilateral presence of Lac in the mesial temporal lobe structures may serve as additional diagnostic clues for lateralization of the epileptogenic zone in MTLE. Metabolic imaging has limited usefulness for differentiation of the hippocampal sclerosis and low-grade intraaxial brain tumor. Presence of significant bilateral metabolic alterations in the mesial temporal lobe structures is associated with worse postoperative seizure control.