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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.

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) 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/).

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.

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.

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.

Huge carbonate rock bodies ranging in age from the Visean (Middle Mississippian/Early Carboniferous) to the Changhsingian (Lopingian/Late Permian) overlie a basaltic basement in the Changning-Menglian Belt, West Yunnan, Southwest China. These carbonates lack intercalations of terrigenous siliciclastic material throughout. These lines of evidence indicate that they formed upon an isolated and continuously subsiding mid-oceanic island (or plateau), probably of hotspot origin. The carbonates are grouped into a shallow-water carbonate platform facies regime observed in the Yutangzhai section and a relatively deep-water carbonate slope facies regime typically represented in the Longdong section. These two facies regimes developed contemporaneously as parts of a carbonate depositional system on and around a mid-oceanic volcanic edifice. The carbonate platform is subdivided into four facies, including platform-margin, shoal, lagoon, and peritidal facies. Along the measured Yutangzhai section of the platform facies regime, the vertical facies succession from the platform-margin facies into inner-platform facies such as the shoal and lagoon facies is recognized. This facies succession is explained as resulting from the progradation of the carbonate platform. Worm tubes occur as a main reef builder in platform-margin facies of the Mississippian. Their occurrence as major constituents in a high-wave-energy reef is peculiar to Carboniferous reef distributions of the world. The occurrences of other reef- and/or mound-building organisms and peritidal dolo-mudstone are almost consistent in timing with those of Panthalassan counterparts such as the Akiyoshi and Omi limestones of Japan, and probably exhibit the worldwide trend.

The lithology of shallow-water carbonates collected from 19 sites on 16 seamounts in six areas of the northwestern Pacific Ocean using the Deep-sea Boring Machine System are described. The areas include the Amami Plateau, Daito Ridge, Oki-Daito Ridge, Urdaneta Plateau, Kyushu-Palau Ridge and Ogasawara Plateau. Chronological constraint is provided by calcareous nannofossil biostratigraphy, planktonic foraminiferal biostratigraphy, larger foraminiferal biostratigraphy and strontium (Sr) isotope stratigraphy. Large amounts of shallow-water carbonates accumulated on the seamounts during the Oligocene, a relatively cool period, whereas limited carbonate deposits formed during the Early Miocene, a relatively warm period. This might indicate that deposition of shallow-water carbonates on seamounts in the northwestern Pacific Ocean was not necessarily controlled by climatic conditions, but was related to volcanism and tectonics that served as foundations for reef/carbonate-platform formation. Remarkable differences in biotic composition exist between Cretaceous and Cenozoic shallow-water carbonates. Late Cretaceous shallow-water carbonates are distinguished by the occurrence of rudists, solenoporacean algae and microencrusters. Middle Eocene to Early Oligocene shallow-water carbonates are dominated by Halimeda or nummulitid and discocyclinid larger foraminifers. Scleractinian corals became common from the Oligocene onward. Nongeniculate coralline algae and larger foraminifers were common to abundant throughout the Eocene to the Pleistocene. The replacement of major carbonate producers in the shallow-water carbonate factory during post-Cretaceous time is in accordance with previous studies and is considered to reflect a shift in seawater chemistry.

The Carboniferous-Permian (Visean-Midian) Omi Limestone in the Akiyoshi Terrane, central Japan is a large carbonate unit developed on a seamount in the Panthalassa Ocean. As the seamount subsided during Carboniferous and Permian time, the carbonate deposition at the top of a seamount was almost continous. Terrigenous siliciclastic sediments are absent, because the seamount was situated in an open-ocean setting. The lower part of this seamount-type limestone records a nearly continuous Carboniferous reef succession.

For a quantitative investigation of eosinophil activation in perennial allergic rhinitis, eosinophil cationic protein (ECP) concentrations were measured by a radioimmunoassay in serum, nasal secretions (ECPWN) and in the supernatant of these nasal secretions (ECPsup) obtained from normal subjects and allergic patients. Levels of ECPWN and ECPsup were higher than that of ECPserum. ECPsup showed a positive correlation with clinical severity, despite the lack of a significant correlation with eosinophilia in nasal smears. ECPWN and ECPserum showed no significant correlation with any of these clinical parameters. There was a weak tendency toward an increase in histamine sensitivity of the nasal mucosa of allergic patients with higher ECPsup although this was not statistically significant. These results suggest accumulation and activation of eosinophils in the allergic nasal mucosa, and also indicate that ECPsup may be a clinical parameter of perennial allergic rhinitis.

制癌剤感受性試験は多数あり, 臨床上癌細胞に対しどの制癌剤が有効であるかの選択は難しく, 患者個人においても癌の種類においても制癌剤の効果は異なる。また, 今までの方法では時間もかかり煩雑であるなどいろいろの問題があった。そこで我々は, 制癌剤感受性試験において簡単・迅速・確実であるクリスタルバイオレット法を取入れ試行し, その有用性について検討してみた。クリスタルバイオレット法と3H-thimidine 法との相関において0.778と強い正の相関を見た。今後このクリスタルバイオレット法を使用した臨床経験を重ね検討するつもりである。

Human sinus mucosal specimens from eight normal individuals were exposed to platelet activating factor (PAF) at concentrations ranging from 10(-6) M to 10(-11) M in a humidified CO2 chamber at 37 degrees C. The mucosal surface of the specimens was recorded on video tapes and magnified 2,500 times on a 19-inch television (TV) monitor. Ciliary activity of each ciliated cell was photoelectrically measured on the TV monitor in real time. PAF induced mucosal damage which resulted in a coarse profile including ciliostasis and exfoliation of epithelial cells. The length of the incubation period in which the initial coarse profile occurred on the mucosal surface inversely correlated with the concentration of exposed PAF ranging from 10(-6) M to 10(-10) M with r = -0.712 (p less than 2 x 10(-4)). Both the control medium and 10(-8) M lysoPAF showed no effect on ciliary activity or mucosal surface alteration even after 24 hr of exposure. Significant ciliary inhibition was noted after 6 hr of exposure to PAF at concentrations of 10(-8) M and 10(-10) M (p less than 0.05). After 10 hr of exposure, significant ciliary inhibition (p less than 0.01) was noted at all concentrations. Inhibition occurred in a time- and dose-dependent manner. The length of the incubation period in which initial ciliostatis occurred and the level of PAF concentration showed an inverse correlation with r = -0.918 (p less than 10(-6)). These results indicate that PAF is cytotoxic to human respiratory mucosa.

To clarify the effects of leukotriene C4 (LTC4) on human ciliated epithelium, ciliary activity of the ethmoid sinus mucosa was measured photoelectrically in tissue culture. At concentrations ranging from 10(-6)M to 10(-9)M, LTC4 showed minimal effects on the ciliated epithelium during the initial 30 minutes of exposure; thereafter, ciliary inhibition was observed in a concentration- and time-dependent manner. Irrigation of the mucosa with culture medium 15 minutes after exposure prevented the LTC4-induced ciliary inhibition. However, irrigation 60 minutes after exposure failed to inhibit 10(-8)M LTC4-induced ciliary dysfunction and mucosal damage. The LTC4-induced ciliary inhibition was blocked in the presence of FPL-55712 and/or Ly-171883, both leukotriene receptor antagonists. L-serine and sodium tetraborate complex (SBC), a gamma-glutamyl transpeptidase (gamma-GTP) inhibitor, also inhibited the LTC4-induced ciliary inhibition. These findings indicate that LTC4 is converted to LTD4 by gamma-GTP during 60 minutes of exposure, and LTC4 itself has minimal direct effects on the ciliated cells.