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Glioblastoma (GBM) is the most common malignant brain tumor and has “immunologically cold” features. Changing GBM to an “immunologically hot” tumor requires a strong trigger that induces initial immune responses in GBM. Allogeneic natural killer cells (NKCs) have gained considerable attention as promising immunotherapeutic tools against cancer, where gene-edited NKCs would result in effective anti-cancer treatment. The present study focused on the immune checkpoint molecule cytokine-inducible SH2-containing protein (CIS) as a critical negative regulator in NKCs. We successfully induced human CIS-deleted NKCs (NK dCIS) by combining our specific human NKC expansion method available for clinical application and genome editing technology. CIS gene-specific guide RNA/Cas9 protein complex suppressed CIS expression in the expanded NKCs with high expansion efficacy. Comprehensive gene expression analysis demonstrated increased expression of 265 genes and decreased expression of 86 genes in the NK dCIS, which were comparatively small changes. Gene set enrichment analysis revealed that the enriched genes were involved in NKC effector functions. Functional analysis revealed that the NK dCIS had increased interferon (IFN)ɤ and tumor necrosis factor (TNF) production. CIS deletion enhanced NKC-mediated apoptosis induction against allogeneic GBM cells and spheroids. Intracranial administration of the allogeneic NKCs prolonged the overall survival of xenograft brain tumor mice. Furthermore, the NK dCIS extended the overall survival of the mice. The findings demonstrated the successful induction of human primary NK dCIS with CRISPR/Cas9 with efficient expansion. CIS deletion enhanced the NKC-mediated anti-tumor effects in allogeneic GBM and could be a promising immunotherapeutic alternative for patients with GBM.

Background. Patients with glioblastoma (GBM) have poor outcomes and novel strategies are needed. Although immunotherapies have been investigated, altering the severe immunosuppressive tumor microenvironment, or so-called “cold tumors”, is difficult. We developed an immunotherapy based on genome-edited natural killer cells (NKCs) with knocking (KO) of checkpoint receptor T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT), which would overcome the GBM immunosuppressive tumor microenvironment. Methods. The GBM TIGIT and TIGIT ligand expression patterns were analyzed with GlioVis and The Human Protein Atlas portal. We generated TIGIT KO human primary NKCs using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) with single guide RNA targeting different genome sites on TIGIT coding exons. The genome-edited NKCs underwent comprehensive microarray gene expression analysis. The anti-GBM activity of the genome-edited NKCs was detected with a 2D adherent model and 3D spheroids derived from allogeneic GBM cells. Results. We successfully obtained TIGIT KO NKCs and effective TIGIT expression KO with unchanged immune checkpoint receptor expression. T7 endonuclease I mutation detection assays demonstrated that the ribonucleoproteins disrupted the intended genome sites. Gene expression analysis demonstrated extremely minimal gene expression pattern changes, such as that for the immune responses, in the TIGIT KO NKCs. Targeting TIGIT KO using CRISPR/Cas9 enhanced NKC anti-tumor activity against the 2D adherent cells and 3D GBM spheroids. Conclusions. Here, we established TIGIT KO human primary NKCs, which demonstrated enhanced anti-tumor activity against GBM cell lines and spheroids. Immunotherapy based on CRISPR/Cas9-edited TIGIT KO NKCs could be a promising GBM therapy.

Irradiated murine induced-pluripotent stem cells (iPSCs) elicit the antitumor response in vivo. However, it is unclear whether human iPSCs would elicit antitumor effects. In the present study, we investigated the capability of human iPSC lysate (iPSL)-pulsed dendritic cells (DCs) (iPSL/DCs) to induce cancer-responsive cytotoxic T lymphocytes (CTLs) in vitro. iPSCs and DCs were induced from peripheral blood mononuclear cells isolated from a human leukocyte antigen (HLA)-A33 homozygous donor. The iPSL was pulsed with immature DCs, which were then stimulated to allow full maturation. The activated DCs were co-cultured with autologous CTLs and their responses to SW48 colorectal carcinoma cells (HLA-A32/A33), T47D breast cancer cells (HLA-A33/A33), and T98G glioblastoma cells (HLA-A02/A02) were tested with enzyme-linked immunospot (ELISPOT) assays. Comprehensive gene expression analysis revealed that the established iPSCs shared numerous tumor-associated antigens with the SW48 and T47D cells. Immunofluorescent analysis demonstrated that the fluorescent-labeled iPSL was captured by the immature DCs within 2 h. iPSL/DCs induced sufficient CTL numbers in 3 weeks for ELISPOT assays, which revealed that the induced CTLs responded to SW48 and T47D cells. Human iPSL/DCs induced cancer-responsive CTLs on HLA-A33-matched cancer cells in vitro and could be a promising universal cancer vaccine for treating and preventing cancer.

To evaluate the safety profile of bis-chloroethyl-nitrosourea (BCNU) wafer implantation after malignant glioma resection with or without ventricular opening (VO).This single-center retrospective study included 66 consecutive patients with BCNU wafer implantation after malignant glioma resection between March 2013 and August 2021. The patients were categorized into two groups based on whether VO occurred during the malignant glioma resection. Fifty-eight patients had glioblastoma, and eight had anaplastic astrocytoma or oligodendroglioma. Forty-eight patients underwent an initial treatment, and 18 underwent recurrent surgeries. Infection, hydrocephalus, subcutaneous fluid collection, chronic subdural hematoma, early seizure after surgery within one month, symptomatic edema surrounding the resected cavity, cyst formation, and postoperative hemorrhage were defined as adverse events (AEs).Thirty-three patients underwent resection with VO, and 33 without. The median survival time was 28 months in the initial treatment group and 11.5 months in the recurrent treatment group. The with and without VO groups had similar median survival times. Postoperative AEs occurred in 7/33 patients (21.2%) with VO and 10/33 (30.3%) without VO, with no difference between them (p = 0.574).This study showed that VO during surgery with BCNU wafer implantation might not influence the occurrence of postoperative AEs. If VO happens, BCNU wafer implantation can be performed safely with accurate closing of the ventricle.

Borehole logs are important geospatial data for understanding subsurface geological and geotechnical conditions. Recently, many borehole logs in urban areas have been open to the public on the Internet in machine-readable file format. However, a few simple methods allow us to construct a voxel model of geological and geotechnical characteristics with high objectivity of the modeling process. Our work aims to develop a simple method that facilitates the use of open borehole logs in geological and geotechnical investigations in urban areas. In this paper, we propose a voxel modeling method of geotechnical data using the machine-readable open borehole logs. The method is designed to construct voxel models of lithofacies and standard penetration test results by stacking a series of horizontal two-dimensional grid data that are created by natural neighbor interpolation using geotechnical data stored in borehole logs at regular elevation intervals. A test calculation to evaluate the proposed method was performed using a large number of borehole logs in the Tokyo metropolitan area, Japan. Results confirmed that the proposed method not only allows us to ensure the objectivity of the modeling process but also provides a good overview of the three-dimensional distribution pattern of the subsurface geotechnical characteristics without any setting of complicated calculation parameters. In conclusion, the proposed method improves the efficiency of geological analyses, such as stratigraphic correlation using borehole logs, and facilitates the use of open borehole logs in urban areas.

Cell-based immunotherapy is categorized as a regenerative therapy under the Regenerative Medicine Safety Act in Japan. Natural killer (NK) cell-based immunotherapy is considered a promising strategy for treating cancer, including glioblastoma (GBM). We previously reported an expansion method for highly purified human peripheral blood-derived NK cells using a cytokine cocktail. Here, we aimed to establish a more efficient NK cell expansion method as compared to our previously reported method.T cell-depleted human peripheral blood mononuclear cells (PBMCs) were isolated from three healthy volunteers. The depleted PBMCs were cultured in the presence of recombinant human interleukin (rhIL)-18 and high-dose rhIL-2 in anti-NKp46 and/or anti-CD16 antibody immobilization settings. After 14 days of expansion, the purity and expansion ratio of CD3-CD56+ NK cells were determined. The cytotoxicity-mediated growth inhibition of T98G cells (an NK activity-sensitive GBM cell line) was evaluated using a non-labeling, impedance-based real-time cell analyzer.Anti-NKp46 stimulation increased the NK cell purity and expansion ratio as compared to the non-antibody-stimulated population. Anti-CD16 stimulation weakly enhanced the NK cell expansion ratio of the non-antibody-stimulated population and enhanced the NK cell purity and expansion ratio of anti-NKp46-stimulated populations. All NK cell-containing populations tested distinctly inhibited T98G cell growth. These effects tended to be enhanced in an NK cell purity-dependent manner. In some cases, anti-CD16 stimulation decreased growth inhibition of T98G cell compared to other conditions despite the comparable NK cell purity.We established a robust large-scale feeder-free expansion system for highly purified human NK cells using a defined cytokine cocktail and anti-NK cell activating receptor antibodies. The expansion system could be feasible for autologous or allogeneic NK cell-based immunotherapy of GBM. Moreover, it is easily controlled under Japanese law on regenerative medicine.

Glioblastoma (GBM) is the most aggressive and malignant primary brain tumor in adults. Despite multimodality treatment involving surgical resection, radiation therapy, chemotherapy, and tumor-treating fields, the median overall survival (OS) after diagnosis is approximately 2 years and the 5-year OS is poor. Considering the poor prognosis, novel treatment strategies are needed, such as immunotherapies, which include chimeric antigen receptor T-cell therapy, immune checkpoint inhibitors, vaccine therapy, and oncolytic virus therapy. However, these therapies have not achieved satisfactory outcomes. One reason for this is that these therapies are mainly based on activating T cells and controlling GBM progression. Natural killer (NK) cell-based immunotherapy involves the new feature of recognizing GBM via differing mechanisms from that of T cell-based immunotherapy. In this review, we focused on NK cell-based immunotherapy as a novel GBM treatment strategy.

Simple Summary Glioblastoma (GBM) is the most aggressive primary malignant brain tumor in adults. Despite standard treatment, including surgery, chemotherapy, and radiotherapy, it is associated with poor survival. Immunotherapy is a promising alternative for patients with GBM. Natural killer (NK) cells are possible promising targets in GBM treatment because of their potent cytotoxic effect. We previously reported that highly activated and ex vivo-expanded NK cells, or genuine induced NK cells (GiNK), exert a greatly cytotoxic effect on GBM cells. In this study, we investigated the potential of NK cell-based immunotherapy for GBM, which we evaluated using an ex vivo three-dimensional GBM cell-derived spheroid model. Our results indicated that the NK cells had an anti-tumor effect on the spheroid models. Our findings could lead to the development of future NK cell-based immunotherapies for GBM. Abstract Glioblastoma (GBM) is the most common and aggressive primary brain tumor, with a dismal prognosis. Natural killer (NK) cells are large granular lymphocytes with natural cytotoxicity against tumor cells, and they should be established for the novel treatment of patients with GBM. We previously reported highly activated, and ex vivo-expanded NK cells derived from human peripheral blood, designated genuine induced NK cells (GiNK), which were induced by specific culture conditions and which exerted a cytotoxic effect on GBM cells via apoptosis. Here, we comprehensively summarize the molecular characteristics, especially focusing on the expression of stem cell markers, extracellular matrix markers, chemokines, chemokine receptors, and NK receptor ligands of spheroids derived from GBM cell lines as compared with that of two-dimensional (2D) adherent GBM cells via microarray. The spheroid had upregulated gene expression of stem cell markers, extracellular matrix markers, chemokines, chemokine receptors, and NK cell inhibitory receptor ligands compared with the 2D adherent GBM cells. Preclinical evaluation of the NK cells was performed via an ex vivo 3D spheroid model derived from GBM cell lines. In the model, the NK cells accumulated and infiltrated around the spheroids and induced GBM cell death. Flow cytometry-based apoptosis detection clearly showed that the NK cells induced GBM cell death via apoptosis. Our findings could provide pivotal information for NK cell-based immunotherapy for patients with GBM.

The Shimosa Group, middle- to late-Pleistocene sedimentary succession, has been the focus of stratigraphic attention because it is beneath the Tokyo metropolitan area of central Japan. It is also of palaeoclimatic significance because it contains important interglacial marine strata of the past 450,000 years. Since the marine strata of the Shimosa Group were formed in the fluvial, estuary, and shallow inner bay known as Palaeo-Tokyo Bay, few occurrences of marine microfossils, make it difficult to quantitatively reconstruct the palaeotemperatures. Here, we extracted long-chain alkenones from the core GS-UR-1 penetrating the Shimosa Group to Marine Isotope Stage (MIS) 11. We found that the alkenone unsaturation ratio appears to reflect the sea surface temperatures (SSTs) of Palaeo-Tokyo Bays formed during MIS 5e, 7e, 9, and 11, which might be recorded around the peak of each interglacial period. The palaeo-SSTs during each interglacial period were 2–3 ℃ higher than the palaeo-SSTs of Tokyo Bay in the pre-industrial era, seemed to reach the similar level as the Holocene thermal maximum. We suggest that the LCA-based proxy, which has not been utilized hitherto in studies on the Shimosa Group, demonstrates its potential to provide palaeoclimatic and stratigraphic information.

Purpose The purpose of this paper is to discuss the development and assessment of learning support environments for academic writing that utilize ICT, such as e-learning and online tutoring, in Japanese higher education. First, the authors introduce the design of an e-learning writing program for the Japanese language and assess whether the program is an effective learning support tool for undergraduates. Second, the authors analyze and assess online tutoring support for academic writing and clarify the merits and disadvantages of online and offline tutoring at writing centers, then suggest instructional strategies by analyzing the writing tutoring process. Design/methodology/approach The authors adopted e-learning goals to assess its effectiveness. The authors asked the participants questions they could answer from five-point scales, conducted a paired t-test, and included a free description-type questionnaire. Then, to assess online tutoring, the 12 students took pre- and post-test questionnaires, after which the authors conducted a Wilcoxon signed rank test. In addition, the authors carried out a Kruskal–Wallis rank sum test in order to confirm differences in satisfaction level and the effectiveness of face-to-face tutoring and online tutoring. Findings By analyzing the pre- and post-test results, the t-test confirmed that the students found the e-learning system to be effective for nurturing academic literacy. This means the system is appropriate as a support tool for nurturing academic writing, especially writing knowledge and rules, and university must provide a comprehensive learning support environment including e-learning. Next, the authors found no statistically significant difference between face-to-face and online tutoring, although some problems with the writing process remained. So online tutoring has opportunity to promote autonomous learning. The research results make it clear that because of writing centers’ potential and their effectiveness in utilizing ICT tools. Originality/value Research findings about academic writing are to improve the tutoring process and writing strategies, such as the use of ICT for academic writing support like e-learning, online tutoring, do not exist. To provide learning opportunity to learners and promote autonomous learning, e-learning and online tutoring are important. For the reasons noted above, it is necessary to provide an alternative writing support environment to students in Japan. Therefore, the authors report on and assess the development of learning support environments for e-learning programs and online tutoring for academic writing at the undergraduate level in Japan.

OBJECTIVELumbar spinal drainage (LSD) during neurosurgery can have an important effect by facilitating a smooth procedure when needed. However, LSD is quite invasive, and the pathology of brain herniation associated with LSD has become known recently. The objective of this study was to determine the risk of postoperative brain herniation after craniotomy with LSD in neurosurgery overall.METHODSIncluded were 239 patients who underwent craniotomy with LSD for various types of neurological diseases between January 2007 and December 2016. The authors performed propensity score matching to establish a proper control group taken from among 1424 patients who underwent craniotomy and met the inclusion criteria during the same period. The incidences of postoperative brain herniation between the patients who underwent craniotomy with LSD (group A, n = 239) and the matched patients who underwent craniotomy without LSD (group B, n = 239) were compared.RESULTSBrain herniation was observed in 24 patients in group A and 8 patients in group B (OR 3.21, 95% CI 1.36–8.46, p = 0.005), but the rate of favorable outcomes was higher in group A (OR 1.79, 95% CI 1.18–2.76, p = 0.005). Of the 24 patients, 18 had uncal herniation, 5 had central herniation, and 1 had uncal and subfalcine herniation; 8 patients with other than subarachnoid hemorrhage were included. Significant differences in the rates of deep approach (OR 5.12, 95% CI 1.8–14.5, p = 0.002) and temporal craniotomy (OR 10.2, 95% CI 2.3–44.8, p = 0.002) were found between the 2 subgroups (those with and those without herniation) in group A. In 5 patients, brain herniation proceeded even after external decompression (ED). Cox regression analysis revealed that the risk of brain herniation related to LSD increased with ED (hazard ratio 3.326, 95% CI 1.491–7.422, p < 0.001). Among all 1424 patients, ED resulted in progression or deterioration of brain herniation more frequently in those who underwent LSD than it did in those who did not undergo LSD (OR 9.127, 95% CI 1.82–62.1, p = 0.004).CONCLUSIONSBrain herniation downward to the tentorial hiatus is more likely to occur after craniotomy with LSD than after craniotomy without LSD. Using a deep approach and craniotomy involving the temporal areas are risk factors for brain herniation related to LSD. Additional ED would aggravate brain herniation after LSD. The risk of brain herniation after placement of a lumbar spinal drain during neurosurgery must be considered even when LSD is essential.

Background/aim We previously established a novel type of epidermal growth factor receptor variant III (EGFRvIII)-specific chimeric antigen receptor (CAR)-expressing natural killer (NK) cell line, designated EvCAR-KHYG-1, which inhibited the growth of glioblastoma (GBM) cells in vitro via apoptosis. Materials and methods We investigated the cytokine-producing effect of EvCAR-KHYG-1 cells on GBM-like cell lines and their antitumour effect using in vivo xenograft assays. Results EvCAR-KHYG-1 cells produced interleukin-2, interferon-γ, and tumour necrosis factor-α on EGFRvIII-expressing U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 cells did not reduce the volume of subcutaneous tumours derived from EGFRvIII-expressing U87MG cells but did reduce tumour cell occupancy. Conclusion EvCAR-KHYG-1 cells led to expression of cellular immunity-related cytokines on EGFRvIII-expressing U87MG in vitro but did not inhibit tumour progression due to the induction of a pseudo progression-like pathological feature. Future studies investigating the effect of different conditions in vivo are required to study the inhibition of tumour progression in GBM.

Glioblastoma (GBM), which is the most common malignant brain tumor, is resistant to standard treatments. Immunotherapy might be a promising alternative for the treatment of this cancer. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. However, the antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells in GBM are limited. The inhibitory effect is induced by the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on GBM cells. In the present study, PD-1-disrupted EvCAR-T cells were established using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). The sgRNA/Cas9 expression vectors designed precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. The PD-1-disrupted EvCAR-T cells had an in vitro growth inhibitory effect on EGFRvIII-expressing GBM cells without altering the T-cell phenotype and the expression of other checkpoint receptors. In the future, the in vivo antitumor effect of this vector should be evaluated in order to determine if it could be applied clinically for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM.

This article describes a method for processing microtremor records from a small-scale seismic array that allows interval-averaged S-wave velocities to be estimated for 10-m depth ranges down to a depth of 30 m. The method was applied to microtremor data obtained in the town of Mashiki, Kumamoto Prefecture, Japan, and the analysis results were evaluated through a comparison with available PS logs and sections obtained by surface-wave methods. It turned out that the interval-averaged S-wave velocity estimates may be subject to errors of up to 20–30% in absolute values, but it was shown that the method can help evaluate relative spatial variations in those S-wave velocities. In view of the simplicity of analysis, the analyser-independent nature of the results and the limitations of analysis accuracy, the interval-averaged S-wave velocity estimation method presented here could be used as an effective tool for the preliminary analysis of microtremor data from small-scale seismic arrays.

Glioblastoma (GBM) is the leading malignant intracranial tumor and is associated with a poor prognosis. Highly purified, activated natural killer (NK) cells, designated as genuine induced NK cells (GiNKs), represent a promising immunotherapy for GBM. We evaluated the anti-tumor effect of GiNKs in association with the programmed death 1(PD-1)/PD-ligand 1 (PD-L1) immune checkpoint pathway. We determined the level of PD-1 expression, a receptor known to down-regulate the immune response against malignancy, on GiNKs. PD-L1 expression on glioma cell lines (GBM-like cell line U87MG, and GBM cell line T98G) was also determined. To evaluate the anti-tumor activity of GiNKs in vivo, we used a xenograft model of subcutaneously implanted U87MG cells in immunocompromised NOG mice. The GiNKs expressed very low levels of PD-1. Although PD-L1 was expressed on U87MG and T98G cells, the expression levels were highly variable. Our xenograft model revealed that the retro-orbital administration of GiNKs and interleukin-2 (IL-2) prolonged the survival of NOG mice bearing subcutaneous U87MG-derived tumors. PD-1 blocking antibodies did not have an additive effect with GiNKs for prolonging survival. GiNKs may represent a promising cell-based immunotherapy for patients with GBM and are minimally affected by the PD-1/PD-L1 immune evasion axis in GBM., 博士（医学）・甲第827号・令和4年3月15日, © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor in adults. Natural Killer (NK) cells are potent cytotoxic effector cells against tumor cells inducing GBM cells; therefore, NK cell based- immunotherapy might be a promising target in GBM. T cell immunoglobulin mucin family member 3 (TIM3), a receptor expressed on NK cells, has been suggested as a marker of dysfunctional NK cells. We established TIM3 knockout in NK cells, using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9). Electroporating of TIM3 exon 2- or exon 5-targeting guide RNA- Cas9 protein complexes (RNPs) inhibited TIM3 expression on NK cells with varying efficacy. T7 endonuclease I mutation detection assays showed that both RNPs disrupted the intended genome sites. The expression of other checkpoint receptors, i.e., programmed cell death 1 (PD1), Lymphocyte-activation gene 3 (LAG3), T cell immunoreceptor with Ig and ITIM domains (TIGIT), and TACTILE (CD96) were unchanged on the TIM3 knockout NK cells. Real time cell growth assays revealed that TIM3 knockout enhanced NK cell-mediated growth inhibition of GBM cells. These results demonstrated that TIM3 knockout enhanced human NK cell mediated cytotoxicity on GBM cells. Future, CRISPR-Cas9 mediated TIM3 knockout in NK cells may prove to be a promising immunotherapeutic alternative in patient with GBM., 博士（医学）・甲第828号・令和4年3月15日, © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

Nitrogen-containing bisphosphonates (N-BPs), which prevent bone resorption, exert direct and γδT cell (GDT)-mediated antitumor effects against several tumor cell types, including glioblastoma (GBM). However, limited information is available regarding the antitumor effects of N-BPs in GBM. Specifically, the antitumor effects of minodronate (MDA), a third-generation N-BP, in GBM are yet unclear. This study aimed to investigate the antitumor effects of MDA in GBM in vitro and in vivo. We performed growth inhibition and apoptosis detection assays using the GBM cell lines U87MG and U138MG. Apoptosis inhibition assays were also conducted. In vivo xenograft assays were performed in highly immunodeficient NOD.Cg-Prkdc(scid) Il2rg(tm1Sug)/Jic mice subcutaneously implanted with U87MG and U138MG cells. Growth inhibition and apoptosis detection assays demonstrated that MDA inhibited GBM cell growth via apoptosis, which was markedly enhanced by ex vivo expanded GDT. A pan-caspase inhibitor, z-VAD-fmk, inhibited MDA-induced U138MG apoptosis and MDA/GDT-induced U87MG and U138MG apoptosis. But z-VAD-fmk increased MDA-induced U87MG apoptosis. MDA/GDT-mediated apoptosis was blocked by the anti-T cell receptor (TCR) Vγ9, mevalonate pathway inhibitor, granzyme B inhibitor, and antitumor necrosis factor (TNF)-α. In vivo xenograft assays showed that combined intraperitoneal administration of MDA/GDT induced antitumor effects on unestablished U87MG-derived subcutaneous tumors. MDA exerted direct and GDT-mediated anti-GBM apoptotic effects in a caspase-dependent manner. GDT recognized MDA-exposed GBM cells via TCRVγ9 and induced apoptosis via granzyme B and TNF-α release. Because MDA elicited anti-GBM effects in synergy with GDT in vivo, a combination of MDA and ex vivo-generated GDT could be an effective treatment in patients with GBM.

Glioblastoma (GBM) is the most common and malignant brain tumor. As GBM is resistant to standard treatment, immunotherapy might be a promising alternative. Chimeric antigen receptor (CAR) is an artificially modified fusion protein that can be engineered to direct the specificity and function of T cells against tumor antigens. The antitumor effects of EGFRvIII-targeting CAR-T (EvCAR-T) cells are limited in GBM, on account of the interaction between programmed cell death protein 1 (PD-1) on activated EvCAR-T cells and its ligands on target tumor cells. Antibodies against PD-1 have shown limitations, so permanent deletion of the PD-1 gene in CAR-T cells might be a promising solution. Therefore, in the present study, PD-1-disrupted EvCAR-T cells were established for the first time with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) gene-editing tool. We determined the disruption efficiency of the human PD-1 gene in EvCAR-T cells and evaluated the antitumor effect of PD-1-disrupted EvCAR-T cells against human GBM cells. The results showed that our designed-sgRNA/Cas9 expression vectors precisely disrupted the target region of PD-1 and inhibited the expression of PD-1 in EvCAR-T cells. Further, the PD-1-disrupted EvCAR-T cells also had a growth inhibitory effect on EGFRvIII-expressing GBM cells. Thus, this study provides a simple, easy, and clinical applicable strategy for inducing PD-1-disrupted EvCAR-T cells, which could potentially be useful for improving the efficacy of EvCAR-T cell-based adoptive immunotherapy for GBM, and even other cancers.

Background Natural killer (NK) cells are considered potential antitumor effector cells. The aim of this study was to establish a novel type of a chimeric antigen receptor (CAR)-NK cell line (CAR-KHYG-1) specific for epidermal growth factor receptor variant III (EGFRvIII)-expressing glioblastoma (GBM) cells and investigate the antitumor effects of EGFRvIII-specific-CAR-expressing KHYG-1 (EvCAR-KHYG-1) in vitro and in vivo. Materials and Methods EvCAR-KHYG-1 was established by lentiviral-based transduction of the EvCAR gene and magnetic bead-based purification of EvCAR-expressing NK cells. The antitumor effects of EvCAR-KHYG-1 were evaluated using growth inhibition and apoptosis detection assays on GBM cell lines (EGFRvIII-expressing and non-expressing U87MG glioma cells). Cytokine production was determined by cytometric beads array. In vivo xenograft assays were performed in NOG mice subcutaneously implanted with EGFRvIII-expressing U87MG (Ev-U87MG). Results EvCAR-KHYG-1 inhibited GBM cell-growth via apoptosis in an EGFRvIII-expression specific manner in vitro. In addition, EvCAR-KHYG-1 produced IL-2, INF-γ and TNF-α on Ev-U87MG cells. In vivo xenograft assays showed that EvCAR-KHYG-1 did not decrease the volume of subcutaneous tumors but inhibited the occupancy of cancer cells in the subcutaneous tumor. Discussion EvCAR-KHYG-1 enhanced antitumor effects and produced Th1-type cytokines on EGFRvIII-expressing GBM cells, but did not inhibit progression of subcutaneous GBM cell-derived tumors to induce a pseudo progression pathological phenotype. Conclusion A novel type CAR-NK cell line was established. In vivo experiments are required to determine ways it can be used to inhibit tumor progression.

Background Recently, 5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX fluorescence was reported to be a useful tool during total surgical resection of high-grade gliomas. However, the labeling efficacy of protoporphyrin IX fluorescence is lower in metastatic brain tumors compared to that in high-grade gliomas, and the mechanism underlying protoporphyrin IX fluorescence in metastatic brain tumors remains unclear. Lung cancer, particularly non-small cell lung cancer (NSCLC), is the most common origin for metastatic brain tumor. Therefore, we investigated the mechanism of protoporphyrin IX fluorescence in NSCLC and associated metastatic brain tumors. Methods Western blotting and quantitative real-time polymerase chain reaction (qRT-PCR) was employed to evaluate the protein and mRNA levels of five transporters and enzymes involved in the porphyrin biosynthesis pathway: peptide transporter 1 (PEPT1), hydroxymethylbilane synthase (HMBS), ferrochelatase (FECH), ATP-binding cassette 2 (ABCG2), and heme oxygenase 1 (HO-1). The correlation between protein, mRNA, and protoporphyrin IX levels in NSCLC cells were evaluated in vitro. Immunohistochemistry was used to determine proteins that played a key role in intraoperative protoporphyrin IX fluorescence in clinical samples from patients with NSCLC and pathologically confirmed metastatic brain tumors. Results A significant correlation between PEPT1 expression and protoporphyrin IX accumulation in vitro was identified by western blotting (P = 0.003) and qRT-PCR (P = 0.04). Immunohistochemistry results indicated that there was a significant difference in PEPT1 between the intraoperative protoporphyrin IX fluorescence-positive and protoporphyrin IX fluorescence-negative groups (P = 0.009). Conclusion Expression of PEPT1 was found to be positively correlated with 5-ALA-induced protoporphyrin IX accumulation detected by photodynamic reaction in metastatic brain tumors originating from NSCLC.

Background/aim Natural killer (NK) cells are considered potential antitumor effector cells. The aim of this study was to establish a novel type of a chimeric antigen receptor (CAR) NK cell line (CAR-KHYG-1) specific for epidermal growth factor receptor variant III (EGFRvIII)-expressing tumors and investigate the anti-tumor activity of EGFRvIII-specific-CAR-KHYG-1 (EvCAR-KHYG-1). Materials and methods EvCAR-KHYG-1 was established by self-inactivated lentiviral-based transduction of the EvCAR gene and magnetic bead-based purification of EvCAR-expressing NK cells. The anti-tumor effects of EvCAR-KHYG-1 were evaluated using growth inhibition and apoptosis detection assays in glioblastoma (GBM) cell lines (EGFRvIII-expressing and non-expressing U87MG). Results The findings demonstrated that EvCAR-KHYG-1 inhibited GBM cell-growth via apoptosis in an EGFRvIII-expressing specific manner. Conclusion This is the first study to establish a CAR NK cell line based on the human NK cell line KHYG-1. Therapy with EvCAR-KHYG-1 may be an effective treatment option for GBM patients.

This study examined the effects of using an e-portfolio system to identify concerns raised during sessions at Kansai University Writing Center. The ratio of sessions whereby tutors asked students about their problems and progress was significantly high during sessions when users dropped in or were first-time repeaters. The results indicate that one of the e-portfolio systems used by Kansai University, TEC book, gathers and shares information about students and the historical data of instructional results, thus promoting the meeting at the beginning of sessions. Since both tutors and students cooperate in these sessions, it is important for tutors to quickly and easily understand students' concerns. E-portfolio systems such as TEC book can be one of the best kinds of support for writing sessions.

This paper is a report on the design of Japanese writing in e-learning, based on the IDEAL model in undergraduate education. Construction of the IDEAL model involves the following steps: Identify problems and opportunities, define goals, explore possible strategies, anticipate outcomes and act, and look back and learn. We identified writing problems by 1) analyzing students' usage history of the writing center and 2) comparing freshmen's report before and after usage of the writing center in the first year. We then defined the problems based on the analyses and explored alternative solutions. To apply the solutions, an e-learning program, comprising 29 lessons, was developed to nurture Japanese writing skills at the undergraduate level. The e-learning program consisted of five categories: 1) "Before writing," 2) "Gathering information," 3) "How to write: sentence structure," 4) "How to write: sentence expressions," 5) "Reflecting on your report." These learning materials were also integrated with tutoring at writing centers and support faculty staff to teach effective writing lessons. Based on these analyses, we developed e-learning materials. This paper follows the process of developing and accessing web-based learning material for Japanese writing. According to the results of the questionnaire, the effectiveness of e-learning was revealed.

Large oncoids (up to 14 cm) and microbialites are abundant in the Artinskian (Lower Permian) section of mid-Panthalassan Akiyoshi atoll carbonates. The oncoids and microbialites consist mainly of a tubular microproblematicum, girvanellid cyanobacterial filaments, microbial micrite crusts, and pore-filling sparry calcite cements. They are surrounded by intraclastic–bioclastic grainstone/rudstone, indicative of moderate- to slightly high-energy subtidal conditions. The microbial community was the primary boundstone-forming organisms on the Akiyoshi atoll during this time. It represents a transitional stage in a mid-Panthalassan reef succession between a cooler-water autotrophic Palaeoaplysina–microencruster community in the Gzhelian–Asselian and a warmer-water heterotrophic calcareous sponge–microencruster community in the Middle Permian. The flourishing mid-Panthalassan microbial community during the late Early Permian is related to enhanced alkalinity, increasing nutrient levels, elevated sea-surface temperatures, and the absence of major reef-building metazoans, which resulted from Gondwanan deglaciation, climatic changes, and a pulse of active volcanism.

Glioblastoma is the leading malignant glioma with a poor prognosis. This study aimed to investigate the antitumor effects of natural killer cells in combination with temozolomide as the standard chemotherapeutic agent for glioblastoma. Using a simple, feeder-less, and chemically defined culture method, we expanded human peripheral blood mononuclear cells and assessed the receptor expression, natural killer cell activity, and regulatory T cell frequency in expanded cells. Next, using the standard human glioblastoma cell lines (temozolomide-sensitive U87MG, temozolomide-resistant T98G, and LN-18), we assessed the ligand expressions of receptors on natural killer cells. Furthermore, the antitumor effects of the combination of the expanded natural killer cells and temozolomide were assessed using growth inhibition assays, apoptosis detection assays, and senescence-associated β-galactosidase activity assays in the glioblastoma cell lines. Novel culture systems were sufficient to attain highly purified (>98%), expanded (>440-fold) CD3−/CD56+ peripheral blood-derived natural killer cells. We designated the expanded population as genuine induced natural killer cells. Genuine induced natural killer cells exhibited a high natural killer activity and low regulatory T cell frequency compared with lymphokine-activated killer cells. Growth inhibition assays revealed that genuine induced natural killer cells inhibited the glioblastoma cell line growth but enhanced temozolomide-induced inhibition effects in U87MG. Apoptosis detection assays revealed that genuine induced natural killer cells induced apoptosis in the glioblastoma cell lines. Furthermore, senescence-associated β-galactosidase activity assays revealed that temozolomide induced senescence in U87MG. Genuine induced natural killer cells induce apoptosis in temozolomide-sensitive and temozolomide-resistant glioblastoma cells and enhances temozolomide-induced antitumor effects in different mechanisms. Hence, the combination of genuine induced natural killer cells and temozolomide may prove to be a promising immunochemotherapeutic approach in patients with glioblastoma if the antitumor effects in vivo can be demonstrated.

Glioblastoma (GBM) is a highly aggressive brain tumor for which novel therapeutic approaches, such as immunotherapy, are urgently needed. Zoledronate (ZOL), an inhibitor of osteoclastic activity, is known to stimulate peripheral blood-derived γδT cells and sensitize tumors to γδT cell-mediated killing. To investigate the feasibility of γδT cell-based immunotherapy for patients with GBM, we focused on the killing of GBM cell lines by γδT cells and the molecular mechanisms involved in these cell-cell interactions. Peripheral blood mononuclear cells were expanded in ZOL and interleukin (IL)-2 for 14 days, and γδT cells were enriched in the expanded cells by the immunomagnetic depletion of αβT cells. Gliomas are resistant to NK cells but susceptible to lymphokine-activated killer cells and some cytotoxic T lymphocytes. When the γδT cell-mediated killing of three GBM cell lines (U87MG, U138MG and A172 cells) and an NK-sensitive leukemia cell line (K562 cells) were tested, 32% U87MG, 15% U138MG, 1% A172, and 50% K562 cells were killed at an effector:target ratio of 5:1. The γδT cell-mediated killing of all three GBM cell lines was significantly enhanced by ZOL and this ZOL-enhanced killing was blocked by an anti-T cell receptor (TcR) antibody. These results indicated that TcR γδ is crucial for the recognition of ZOL-treated GBM cells by γδT cells. Since the low level killing of GBM cells by the γδT cells was enhanced by ZOL, γδT cell-targeting therapy in combination with ZOL treatment could be effective for patients with GBM.

Middle Permian lagoonal bioherms are described from mid-Panthalassan atoll carbonates (Akiyoshi Limestone) in the Akiyoshi accretionary complex, SW Japan. The bioherms are composed of frame-building sphinctozoan and inozoan sponges, together with encrusting organisms, including chaetetids, Shamovella, calcareous red algae, and other microbes. Of them, sphinctozoan sponges, Shamovella, and microbes (forming microbialites) are the most dominant. Chaetetids, which were predominant in the Early Pennsylvanian but rapidly decreased in the late Moscovian, are also common in these bioherms. Archaeolithoporella and bryozoans are rare, probably due to the low-energy lagoonal environment. This sponge–microencruster community replaced the Gzhelian–Asselian Palaeoaplysina–microencruster community on the Akiyoshi atolls during the Sakmarian–Kubergandian interval. This drastic change in the reef-building community type almost coincides with the rapid deglaciation of the Gondwanan ice sheet and superplume activity beneath the mid-Panthalassan ocean. The prosperity of calcareous sponges was possibly controlled by environmental factors such as warming and eutrophication after the Gondwanan glaciation. Copyright © 2012 John Wiley & Sons, Ltd.

A Palaeoaplysina–microencruster reef community is recognized in the Gzhelian–Asselian interval of the Taishaku and Akiyoshi limestones in SW Japan, which represent accreted Panthalassan atoll carbonates. In a reef-core area of the atoll, the alga Palaeoaplysina acted as a framebuilder and was associated with various binders (e.g., Tubiphytes, Archaeolithoporella, cystoporate bryozoans, and microbialites) and sediment-bafflers such as fenestrate and cryptostomate bryozoans and phylloid algae. In a subtidal environment of the back-reef area, Palaeoaplysina and phylloid algae flourished as sediment-bafflers. A microbial community, including Tubiphytes and sessile foraminifers, was predominant in a very shallow, peritidal environment of the back-reef area. Palaeoaplysina was distributed mainly along the northern margin of Pangea and is regarded as a boreal element. During Gzhelian–Asselian time, its distribution extended to the tropical or subtropical Panthalassa Ocean due to global cooling. On the Akiyoshi atolls, the Palaeoaplysina–microencruster community was succeeded by a sponge–microencruster community in the late Early Permian. The timing of this biotic turnover is similar to that of a change in climate from icehouse to greenhouse conditions, and coincides with superplume activity beneath the mid-Panthalassa Ocean.

新潟県糸魚川市及びその周辺には日本有数のヒスイ産地があり，また代表的な構造線である糸魚川－静岡構造線が分布する．糸魚川市はこれらの多種多様な地質をもとに一般市民への普及活動とジオパークの整備を行ってきた．2009年8月には北海道洞爺湖有珠山，九州島原半島と並んで日本で初めて世界ジオパークに認定された．一方，2010年春には独立行政法人産業技術研究所より5万分の1地質図幅「小滝」（長森ほか，2010）が発行され，糸魚川ジオパーク内の地質に関する多くの新知見が明らかになってきた．本コースではその中から，糸魚川－静岡構造線の西側に分布する基盤岩類である，青海ヒスイ産地，約300 Ma 蓮華変成岩，秋吉帯に属する石炭系～ペルム系青海石灰岩，舞鶴帯に属するペルム系虫川層と琴沢火成岩類及び倉谷変成岩類，舞鶴帯と秋吉帯の境界断層，そして糸魚川－静岡構造線露頭，さらに東側のリフティングに伴う火成活動で形成された中新世枕状溶岩を観察する．

This paper traces late Palaeozoic second-order sea-level change based on the stratigraphy of the Akiyoshi Limestone, an accreted Carboniferous-Permian Panthalassan atoll carbonate succession in SW Japan. The estimated subsidence of a volcanic edifice and the variable rate of carbonate accumulation reveal the long-term sea-level history of the late Palaeozoic. During Bashkirian time, just after the lowstand stage at the mid-Carboniferous boundary, a slow progressive long-term sea-level rise began. This sea-level rise then increased greatly during Moscovian time. The following Kasimovian to Asselian interval represents a stable highstand stage. Beginning in the Sakmarian, sea level fell slowly and became stable in the Yakhtashian (= Artinskian). This stable sea level was maintained into the Midian (= Capitanian), although a small-scale sea-level rise of approximately 55–70 m is recognized in the Murgabian (= Wordian). The most noteworthy aspect of this change of sea level is the rapid sea-level rise during the Moscovian that created a very large accommodation space and resulted in accumulation of a thick carbonate succession. The eustatic rise from the earliest Bashkirian lowstand to the latest Moscovian highstand had an amplitude of approximately 230–240 m. Such a large-scale eustatic rise in the long-term sea-level change would most likely be caused by greater uplift of the ocean floor along the mid-oceanic ridges, as a result of an increase in the production of oceanic crust during that time.