Published papers

We previously reported that implantation of dendritic cells (DCs) into the injured site activates neural stem/progenitor cells (NSPCs) and promotes functional recovery after spinal cord injury (SCI) in mice. Working toward clinical application of DC therapy for SCI, we analyzed whether DCs promote functional recovery after SCI in a non-human primate, the common marmoset (CM). CMs are usually born as dizygotic twins. They are thus natural bone marrow and peripheral blood chimeras due to sharing of the placental circulation between dizygotic twins, leading to functional immune tolerance. In this study, to identify adequate CM donor-and-host pairs, mixed leukocyte reaction (MLR) assays were performed. Then, CM-DCs were generated from the bone marrow of the twin selected to be donor and transplanted into the injured site of the spinal cord of the other twin selected to be host, 7 days after injury. Histological analyses revealed fewer areas of demyelination around the injured site in DC-treated CMs than in controls. Immunohistochemical analysis showed that more motor neurons and corticospinal tracts were preserved after SCI in DC-treated CMs. Motor functions were evaluated using three different behavior tests and earlier functional recovery was observed in DC-treated CMs. These results suggest DC therapy to possibly be beneficial in primates with SCI and that this treatment has potential for clinical application. © 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society.

Progress in stem cell research reveals cancer stem cells to be present in a variety of malignant tumors. Since they exhibit resistance to anticancer drugs and radiotherapy, analysis of their properties has been rapidly carried forward as an important target for the treatment of intractable malignancies, including brain tumors. In fact, brain cancer stem cells (BCSCs) have been isolated from brain tumor tissue and brain tumor cell lines by using neural stem cell culture methods and isolation methods for side population (SP) cells, which have high drug-efflux capacity. Although the analysis of the properties of BCSCs is the most important to developing methods in treating BCSCs, the absence of BCSC purification methods should be remedied by taking it up as an important research task in the immediate future. Thus far, there are no effective treatment methods for BCSCs, and several treatment methods have been proposed based on the cell biology characteristics of BCSCs. In this article, I outline potential treatment methods damaging treatment-resistant BCSCs, including immunotherapy which is currently a topic of our research.

Previously, we demonstrated that implanted dendritic cells (DCs) in the injured spinal cord of adult mice exert a neurotrophic effect, resulting in the activation of endogenous neural stem/progenitor cells (NSPCs) and neurogenesis. Granulocyte-macrophage colony stimulating factor (GM-CSF), which is an essential cytokine for the generation of DCs from haematopoietic progenitor cells, has been shown to be beneficial for the treatment of spinal cord injury (SCI). In the present study, to evaluate the mechanisms underlying this therapeutic efficacy of GM-CSF, we investigated the effects of GM-CSF on the DC-like cells and NSPCs in the injured spinal cord. When GM-CSF was injected into the injured spinal cords of mice, the numbers of DC-like cells and activated microglia/macrophages around the lesion site increased, accompanied by an increase in BDNF expression. A significant increase in endogenous NSPCs was observed around the lesion site in the GM-CSF-treated mice compared with that in the controls. A neurosphere forming assay revealed that GM-CSF also induced the proliferation of NSPCs in vitro. Moreover, injection of GM-CSF into the lesion immediately after the SCI resulted in early recovery of the locomotor function of the injured mice. In conclusion, GM-CSF activated DC-like cells and NSPCs in the injured spinal cord, which was probably involved in its beneficial effects in cases of spinal cord injury. © 2009 Elsevier Ireland Ltd and the Japan Neuroscience Society.

The current basic goals in glioma surgery are radical tumor resection without triggering the development of new neurological deficits. Although complete removal of malignant gliomas is extremely difficult because of the tumors' infiltrative characteristics, gross total resection of the tumor is known to be associated with improved outcome in the patients. To enable safe and radical resection of malignant gliomas, especially those adjacent to eloquent brain areas, rapid progress has been made in the development of operative support techniques; e.g., navigation systems to provide information about the anatomical and functional locations in the brain and a fluorescence imaging technique for differentiating brain tumors from normal brain tissue. These intraoperative imaging techniques in glioma surgery have improved the functional outcomes of glioma patients.

We have previously reported that the transplantation of dendritic cells (DCs) brings about functional recovery after spinal cord injury in mice through the activation of endogenous microglia/macrophages and neural stem/progenitor cells. In this study, the effect of interleukin-12 (IL-12), which is secreted from DCs, was evaluated for the treatment of spinal cord injury in mice. Administration of IL-12 into the injured site significantly increased the number of activated microglia/macrophages and DCs as well as the expression of brain-derived neurotrophic factor surrounding the lesion site. Immunohistochemical analyses showed that de novo neurogenesis and remyelination were induced by IL-12 treatment. Furthermore, an open field test using Basso-Beattie-Brenham scoring revealed a significant improvement of locomotor function in mice treated with IL-12. These results suggest that IL-12 administration into the injured spinal cord results in a functional recovery through the activation of microglia/macrophages and DCs. © 2008 Wiley-Liss, Inc.

We previously reported identifying SOX6 as a glioma antigen by serological screening using a testis cDNA library. Its preferential expression and frequent IgG responses in glioma patients indicate that SOX6 may be a useful target for immunotherapy. To examine whether cytotoxic T-lymphocyte (CTL) responses specific for SOX6 to destroy glioma can be generated in vivo, we treated glioma-bearing mice by vaccination with a plasmid DNA encoding murine full-length SOX6 protein. Following SOX6-DNA vaccination, CTLs specific for SOX6-expressing glioma cells were induced, while normal autologous-cells that had restrictedly expressed SOX6 during embryogenesis were not destroyed. Furthermore, DNA vaccination with SOX6 exerted protective and therapeutic antitumor responses in the glioma-bearing mice. This antitumor activity was abrogated by the depletion of CD4 positive T cells and/or CD8 positive T cells. These results suggest that the SOX6 protein has multiple CTL and helper epitopes to induce antitumor activity and the effectiveness of SOX6-DNA vaccine for the prevention and treatment of glioma. © 2008 Wiley-Liss, Inc.

Dendritic cells (DCs) have important functions as modulators of immune responses, and their ability to activate T cells is of great value in cancer immunotherapy. The isolation of DCs from the peripheral blood of rhesus and African green monkeys has been reported, but the immune system in the common marmoset remains poorly characterized, although it offers many potential advantages for preclinical studies. In the present study, we devised methods, based on techniques developed for mouse and human DC preparation, for isolating DCs from three major tissue sources in the common marmoset: bone marrow (BM), spleen and peripheral blood. Each set of separated cells was analysed using the cell surface DC-associated markers CD11c, CD80, CD83, CD86 and human leucocyte antigen (HLA)-DR, all of which are antibodies against human antigens, and the cells were further characterized both functionally and morphologically as antigen-presenting cells. BM proved to be an excellent cell source for the isolation of DCs intended for preclinical studies on cell therapy, for which large quantities of cells are required. In the BM-derived CD11c+ cell population, cells exhibiting the characteristic features of DCs were enriched, with the typical DC morphology and the abilities to undergo endocytosis, to secrete interleukin (IL)-12, and to stimulate Xenogenic T cells. Moreover, BM-derived DCs produced the neurotrophic factor NT-3, which is also found in murine splenic DCs. These results suggest that BM-derived DCs from the common marmoset may be useful for biological analysis and for preclinical studies on cell therapy for central nervous system diseases and cancer. © 2007 Blackwell Publishing Ltd.

To identify neuron-specific genes, we performed gene expression profiling, cDNA microarray and in silico ESTs (expressed sequence tags) analyses. We identified a human neuron-specific gene, KIAA1110 (homologue of rat synArfGEF (Po)), that is a member of the guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF). RT-PCR analysis showed that the KIAA1110 gene was expressed specifically in the brain among adult human tissues, whereas no apparent expression was observed in immature neural tissues/cells, such as fetal brain, glioma tissues/cells, and neural stem/precursor cells (NSPCs). The KIAA1110 protein was shown to be expressed in mature neurons but not in undifferentiated NSPCs. Immunohistochemical analysis also showed that KIAA1110 was expressed in neurons of the human adult cerebral cortex. Furthermore, the pull-down assay revealed that KIAA1110 has a GEF activity toward ARF1 that regulates transport along the secretion pathway. These results suggest that KIAA1110 is expressed specifically in mature neurons and may play an important role in the secretion pathway as a GEF for ARF1. © 2007 Elsevier Inc. All rights reserved.

We previously reported to identify SOX5 as a glioma antigen by serological screening using a testis cDNA library. The present study was designed to analyze SOXS expression, its immunoreactivity, and the correlation between SOX5 IgG responses and clinical features in glioma patients to evaluate the possibility of its use as a diagnostic marker. Quantitative RT-PCR and Western blot analysis revealed that SOX5 was expressed in glioma tissues, but not in normal adult tissues, except in the testis. An immunohistochemical analysis showed that SOX5 was expressed in glioma cells, but only a few SOX5-positive cells were detected in non-neoplastic tissues from the cerebral cortex. IgG antibodies against SOX5 were detected in sera from 8 of the 27 glioma patients (27.6%), 0 of the 14 patients with other brain diseases (0%), 1 of the 54 other cancer patients (1.9%) and 1 of the 37 healthy individuals (2.7%). Patients with glioblastotna (GBM) who showed IgG responses against SOX5 exhibited significantly better survival periods than GBM patients without SOX5 antibodies. In summary, SOX5 is aberrantly expressed in glioma and can be recognized as a glioma antigen using IgGs from the sera of glioma patients. Furthermore, there is a statistically significant correlation between the presence of SOX5 IgGs and survival in GBM patients, suggesting that the glioma antigen SOX5 may be useful not only as a diagnostic marker, but also as a prognostic marker in glioma patients. © 2007 Wiley-Liss, Inc.

Purpose: We developed an effective immunotherapy, which could induce antitumor immune responses against shared and unique tumor antigens expressed in autologous tumors. Experimental Design: Intratumoral administration of dendritic cells is one of the individualized immunotherapies; however, the antitumor activity is relatively weak. In this study, we attempted to enhance the antitumor efficacy of the i.t. dendritic cell administration by combining dendritic cells stimulated with Bacillus Calmette-Guerin cell wall skeleton (BCG-CWS) additionally with cryoablative pretreatment of tumors and analyzed the therapeutic mechanisms. Results: These two modifications (cryoablation of tumors and BCG-CWS stimulation of dendritic cells) significantly increases the antitumor effect on both the treated tumor and the untreated tumor, which was distant at the opposite side, in a bilateral s.c. murine CT26 colon cancer model. Further analysis of the augmented antitumor effects revealed that the cryoablative pretreatment enhances the uptake of tumor antigens by the introduced dendritic cells, resulting in the induction of tumor-specific CD8+ T cells responsible for the in vivo tumor regression of both treated and remote untreated tumors. This novel combination i.t. dendritic cell immunotherapy was effective against well-established large tumors. The antitumor efficacy was further enhanced by depletion of CD4+CD25 +FoxP3+ regulatory T cells. Conclusions: This novel dendritic cell immunotherapy with i.t. administration of BCG-CWS-treated dendritic cells following tumor cryoablation could be used for the therapy of cancer patients with multiple metastases. © 2006 American Association for Cancer Research.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was found to promote collateral flow in patients with coronary artery disease and also to induce arteriogenesis in a rat hypoperfusion brain model. Activated macrophages have been shown to induce vascular proliferation and play an important role in ischemic stroke. In this study, we examined the therapeutic effect of GM-CSF on the ischemic brain by activating microglia/macrophages. Rats were subjected to 1-h intraluminal middle cerebral artery occlusion (MCAO) and received an intracarotid injection of GM-CSF (5 ng) or saline immediately after reperfusion. Infarct volume, neurological function and histological findings were assessed 48 h later. An intracarotid injection of GM-CSF reduced the infarct volume and improved neurological function at 48 h after reperfusion. Histological analysis revealed that the number of activated microglia/macrophages to be increased and the number of apoptotic cells to be decreased in the area of the penumbra. These results suggest that intracarotid injection of GM-CSF may have a therapeutic effect on brain ischemia via activation of microglia/macrophages. © 2006 Elsevier B.V. All rights reserved.

Despite several ongoing clinical trials of immunotherapies against glioma, few glioma-specifk antigens recognized by cytotoxic T lymphocytes (CTLs) have been identified. We recently demonstrated that intratumoral inoculation with herpes simplex virus (HSV) as a cancer vaccine activates tumor-specific CTLs. To identify glioma antigens recognized by CTLs, we used the HSV cancer vaccine to vaccinate mice harboring a syngeneic mouse glioma cell line, GL261. From the splenocytes of the immunized mice, we generated an H-2Db-restricted CTL line, GCL-1, that was specific for GL261. Then, a cDNA expression library generated from GL261 was screened with GCL-1, and a new gene encoding glioma antigen, GARC-1, was isolated. Sequence analysis revealed that the GARC-1 gene isolated from GL261 had a point mutation causing an amino acid change (Asp to Asn at position 81). T-cell epitope analysis revealed that the mutated peptide GARC-177-85 (AALLNKLYA) but not the wild-type peptide (AALLDKLYA), was recognized by GCL-1. These results suggest that HSV cancer vaccination may be a useful method for inducing tumor-specific CTLs and identifying tumor antigens. Furthermore, this GL261/GARC-1 murine glioma model may be useful for the development of immunotherapy for brain tumors. © 2005 Wiley-Liss, Inc.

Introduction: Dural and anterior cranial base reconstruction is essential in the surgical resection of a craniofacial tumor that extends from the paranasal sinuses to the subdural space. Watertight reconstruction of vascularized tissue is essential to prevent postoperative liquorrhea, especially under conditions that prevent wound healing (e.g., postoperative irradiation therapy).Method: We successfully treated two cases of olfactory neuroblastoma by endoscopic-assisted craniotomy with primary dural closure and anterior cranial base reconstruction using a multi-layered flap technique. Dural defects were closed using temporal fascia or fascia lata in a conventional fashion, immediately after detaching the subdural tumor, in order to isolate and prevent contamination of subdural components and cerebrospinal fluid (CSF) from the tumor and nasal sinuses. Tumor removal and anterior cranial base reconstruction were performed without any concern of CSF contamination after dural closure by craniotomy and endoscopic endonasal approach (EEA). Vascularized pericranial flaps (PCF) and nasoseptal flaps (NSF) were used simultaneously as doubled-over layers for reconstruction.Results: The tumor was completely removed macroscopically and the anterior cranial base was reconstructed in both cases. CSF leak and postoperative meningitis were absent. Postoperative and irradiation therapy courses were successful and uneventful.Conclusions: This multi-layered anterior cranial base reconstruction consisted of three layers: a fascia for dural plasty and double-layered PCF and NSF. This surgical reconstruction technique is suitable to treat craniofacial tumors extending into the subdural space through the anterior cranial base dura mater.

The surgical results of 18 cases of clival/upper cervical chordoma treated in the last decade via the endoscopic endonasal approach (EEA, 9 cases) and the transoral-transpalatal approach (TO-TPA, 9 cases) were compared. Each group showed the same incidence of subdural invasion, with 5 cases each. The superior (frontal base) and lateral surgical fields were wider by EEA, but the inferior view lower than the cranio-vertebral junction (CVJ) was wider by TO-TPA. Gross total removal was achieved in 3 cases in the EEA group, but in only 1 case in the TO-TPA group. Differences in radicality might be due to the extent of the lateral and subdural overview. However for large tumors extending below the CVJ, TO-TPA was the only viable approach for surgical removal. Surgical complications were higher in the EEA (4 cases) than the TO-TPA group (1 case), and were mainly caused by aggressive management of subdural invasion in the EEA group. Post-operative oral intake was earlier and the operative time was shorter in the EEA group. The surgical results were more radical and less invasive in the EEA group than the TO-TPA group. However in tumors extending below the CVJ, the surgical field in EEA was limited, indicating the need to use the transoral route or a combination of routes. A higher complication rate following subdural management was a negative factor that requires improvement in the EEA group and two-staged EEA followed by a transcranial approach may be considered for the cases with subdural invasion.

The aim of this study was to analyze the bacterial flora in the nasal cavity and sphenoid sinus and evaluate the sensitivity of these bacteria to antibiotics that can be used to prevent postoperative meningitis. Bacteria of the preoperative nasal cavity and intraoperative sphenoid sinus mucosa were cultured and analyzed in 40 patients (20 men and 20 women; mean age, 52.2 years) who underwent endoscopic transsphenoidal surgery. The sensitivity of these bacteria to cephalosporin, a representative prophylactic antibiotic, was examined. Staphylococcus epidermidis was the most frequently detected species in both spaces; 24 (38.7%) of 62 isolates in the nasal cavity and 26 (37.1%) of 70 isolates in the sphenoid sinus. In contrast, Corynebacterium species were found mainly in the nasal cavity, and anaerobic bacteria were found only in the sphenoid sinus. Bacteria that were resistant to cephalosporin were found in the nasal cavity in 3.2% of patients and in the sphenoid sinus in 20% of patients. In conclusion, the composition of bacterial flora, including bacteria that are resistant to prophylactic antibiotics, differs between the nasal cavity and the sphenoid sinus.

BACKGROUND: A cDNA library made from 2 glioma cell lines, U87MG and T98G, was screened by serological identification of antigens by recombinant cDNA expression (SEREX) using serum from a glioblastoma patient. Elongation factor Tu GTP binding domain containing protein 1 (EFTUD1), which is required for ribosome biogenesis, was identified. A cancer microarray database showed overexpression of EFTUD1 in gliomas, suggesting that EFTUD1 is a candidate molecular target for gliomas.METHODS: EFTUD1 expression in glioma cell lines and glioma tissue was assessed by Western blot, quantitative PCR, and immunohistochemistry. The effect on ribosome biogenesis, cell growth, cell cycle, and induction of apoptosis and autophagy in glioma cells during the downregulation of EFTUD1 was investigated. To reveal the role of autophagy, the autophagy-blocker, chloroquine (CQ), was used in glioma cells downregulating EFTUD1. The effect of combining CQ with EFTUD1 inhibition in glioma cells was analyzed.RESULTS: EFTUD1 expression in glioma cell lines and tissue was higher than in normal brain tissue. Downregulating EFTUD1 induced G1 cell-cycle arrest and apoptosis, leading to reduced glioma cell proliferation. The mechanism underlying this antitumor effect was impaired ribosome biogenesis via EFTUD1 inhibition. Additionally, protective autophagy was induced by glioma cells as an adaptive response to EFTUD1 inhibition. The antitumor effect induced by the combined treatment was significantly higher than that of either EFTUD1 inhibition or CQ alone.CONCLUSION: These results suggest that EFTUD1 represents a novel therapeutic target and that the combination of EFTUD1 inhibition with autophagy blockade may be effective in the treatment of gliomas.

Purpose Pilocytic astrocytomas (PAs) are the most common brain tumor in children and typically have an excellent prognosis. However, some PAs show histologically anaplastic features. It is reported that PAs with anaplastic features often need the postoperative radiation and chemotherapy due to aggressiveness such as early local recurrence and dissemination. We describe an interesting case of primary anaplastic PA with good clinical course in the long-term. Methods A 10-year-old man presented with worsening headache and vomiting. Magnetic resonance imaging (MRI) showed a large cystic tumor with contrast-enhanced solid component in a right occipital lobe. Magnetic resonance spectroscopy (MRS) showed the decrease of N-acetylaspartate (NAA) and the increase of choline and lipids, which suggested the malignancy. Results The patient was operated with an occipital lobectomy. The tumor was incompletely resected due to the deep invasion to the inner wall of lateral ventricle. Pathological diagnosis was a pilocystic astrocytoma with anaplastic features. Although aggressive features were suspected from magnetic resonance spectroscopy and pathological findings, the remnant tumor showed no recurrence for 8 years without any postoperative treatments. Conclusion PAs could exhibit variable behavior, and careful managements including wait-and-scan should be considered, because adjuvant therapies may cause child's growth disorder and malignant transformation. © 2014 Springer-Verlag Berlin Heidelberg.

Macrophage migration inhibitory factor (MIF) has important roles in supporting the proliferation and/or survival of murine neural stem/progenitor cells (NSPCs), but downstream effectors remain unknown. We show here that MIF robustly increases the expression of Sox6 in NSPCs in vitro. During neural development, Sox6 is expressed in the ventricular zone of the ganglionic eminence (GE) of mouse brains at embryonic day 14.5 (E14.5), cultured NSPCs from E14.5 GE, and NSPCs in the subventricular zone (SVZ) around the lateral ventricle (LV) of the adult mouse forebrain. Retroviral overexpression of Sox6 in NSPCs increases the number of primary and secondary neurospheres and inhibits cell differentiation. This effect is accompanied with increased expression of Hes1 and Bcl-2 and Akt phosphorylation, thus suggesting a role for Sox6 in promoting cell survival and/or self-renewal ability. Constitutive activation of the transcription factor Stat3 results in up-regulation of Sox6 expression and chromatin immunoprecipitation analysis showed that MIF increases Stat3 binding to the Sox6 promoter in NSPCs, indicating that Stat3 stimulates Sox6 expression downstream of MIF. Finally, the ability of MIF to increase the number of primary and secondary neurospheres is inhibited by Sox6 gene silencing. Collectively, our data identify Sox6 as an important downstream effector of MIF signaling in stemness maintenance of NSPCs. © 2013 Ohta et al.

Oligoastrocytoma preferentially arises in the cerebral hemisphere, and a cerebellar location is unusual. We report the case of a 35-year-old woman with an aggressive cerebellar tumor histopathologically diagnosed as oligoastrocytoma World Health Organization (WHO) grade II. After partial removal of the tumor, she underwent concomitant temozolomide (TMZ) therapy with local irradiation followed by additional TMZ monotherapy. However, her symptoms gradually worsened, and chronological magnetic resonance imaging showed remarkable tumor enlargement. In accordance with the aggressive clinical course, unfavorable genetic characteristics such as the gain of the entire chromosome 7, loss of 9p, absence of 1p/19q codeletion, absence of methylation of the O6- methylguanine-deoxyribonucleic acid methyltransferase promoter, and absence of the isocitrate dehydrogenase-1 mutation were observed. The present case illustrates that these molecular characteristics represent the biological features of gliomas more closely than the histopathological diagnosis and may also suggest that infratentorial gliomas arise through a distinct tumorigenic pathway from their supratentorial counterparts. © The Japan Society of Brain Tumor Pathology 2012.