Targeting hemoglobin receptors IsdH and IsdB of Staphylococcus aureus with a single VHH antibody inhibits bacterial growth.

Methicillin-resistant Staphylococcus aureus, or MRSA, is one of the major causative agents of hospital-acquired infections worldwide. Novel antimicrobial strategies efficient against antibiotic-resistant strains are necessary and not only against S. aureus. Among those, strategies that aim at blocking or dismantling proteins involved in the acquisition of essential nutrients, helping the bacteria to colonize the host, are intensively studied. A major route for S. aureus to acquire iron from the host organism is the Isd (iron surface determinant) system. In particular, the hemoglobin receptors IsdH and IsdB located on the surface of the bacterium are necessary to acquire the heme moiety containing iron, making them a plausible antibacterial target. Herein, we obtained an antibody of camelid origin that blocked heme acquisition. We determined that the antibody recognized the heme-binding pocket of both IsdH and IsdB with nanomolar order affinity through its second and third complementary-determining regions. The mechanism explaining the inhibition of acquisition of heme in vitro could be described as a competitive process in which the complementary-determining region 3 from the antibody blocked the acquisition of heme by the bacterial receptor. Moreover, this antibody markedly reduced the growth of three different pathogenic strains of MRSA. Collectively, our results highlight a mechanism for inhibiting nutrient uptake as an antibacterial strategy against MRSA.

Anti-InlA single-domain antibodies that inhibit the cell invasion of Listeria monocytogenes.

Listeriosis, caused by infection with Listeria monocytogenes, is a severe disease with a high mortality rate. The L. monocytogenes virulence factor, internalin family protein InlA, which binds to the host receptor E-cadherin, is necessary to invade host cells. Here, we isolated two single-domain antibodies (VHHs) that bind to InlA with picomolar affinities from an alpaca immune library using the phage display method. These InlA-specific VHHs inhibited the binding of InlA to the extracellular domains of E-cadherin in vitro as shown by biophysical interaction analysis. Furthermore, we determined that the VHHs inhibited the invasion of L. monocytogenes into host cells in culture. High-resolution X-ray structure analyses of the complexes of VHHs with InlA revealed that the VHHs bind to the same binding site as E-cadherin against InlA. We conclude that these VHHs have the potential for use as drugs to treat listeriosis.

Simple tape-stripping method for highly reliable and quantitative analysis of skin microbiome.

The composition of human skin microbiome profoundly impacts host skin health and disease. However, the relationship between skin homeostasis or the development of skin diseases and daily changes in skin microbial composition is poorly understood. Longitudinal samplings at more frequent intervals would address this issue, while conventional sampling methods have technical difficulties, leading to limitations in sampling opportunities. Here, we developed a simple and stable tape-stripping method regardless of the operator's skill. Our method enables skin microbial sampling within 30 seconds and taking multiple skin microbial samples from the same body site. The amount of microbial DNA among multiple sampling sites could be measured within 13.5%. The sequencing results of multiple sampling showed high consistency, Pearson's correlation coefficient between multiple samples of 0.98. Furthermore, these results were comparable to those collected by the conventional swabbing method. These results demonstrate that our tape-stripping method enables simple microbiome collection and highly reliable quantitative skin microbiome analysis. These features of our method would lead to a further understanding of skin disease development or diagnosis of skin conditions in clinical research by increasing the opportunities for microbial sampling.

Mitochondrial Calcium Uniporter (MCU) is Involved in an Ischemic Postconditioning Effect Against Ischemic Reperfusion Brain Injury in Mice.

The phenomenon of ischemic postconditioning (PostC) is known to be neuroprotective against ischemic reperfusion (I/R) injury. One of the key processes in PostC is the opening of the mitochondrial ATP-dependent potassium (mito-KATP) channel and depolarization of the mitochondrial membrane, triggering the release of calcium ions from mitochondria through low-conductance opening of the mitochondrial permeability transition pore. Mitochondrial calcium uniporter (MCU) is known as a highly sensitive transporter for the uptake of Ca2+ present on the inner mitochondrial membrane. The MCU has attracted attention as a new target for treatment in diseases, such as neurodegenerative diseases, cancer, and ischemic stroke. We considered that the MCU may be involved in PostC and trigger its mechanisms. This research used the whole-cell patch-clamp technique on hippocampal CA1 pyramidal cells from C57BL mice and measured changes in spontaneous excitatory post-synaptic currents (sEPSCs), intracellular Ca2+ concentration, mitochondrial membrane potential, and N-methyl-D-aspartate receptor (NMDAR) currents under inhibition of MCU by ruthenium red 265 (Ru265) in PostC. Inhibition of MCU increased the occurrence of sEPSCs (p = 0.014), NMDAR currents (p < 0.001), intracellular Ca2+ concentration (p < 0.001), and dead cells (p < 0.001) significantly after reperfusion, reflecting removal of the neuroprotective effects in PostC. Moreover, mitochondrial depolarization in PostC with Ru265 was weakened, compared to PostC (p = 0.004). These results suggest that MCU affects mitochondrial depolarization in PostC to suppress NMDAR over-activation and prevent elevation of intracellular Ca2+ concentrations against I/R injury.

Pre-treatment systemic inflammation response index and systemic immune inflammation in patients with primary central nerve system lymphoma as a useful prognostic indicator.

PURPOSE: The systemic inflammation response index (SIRI) and systemic immune-inflammation index (SII) are based on neutrophil, monocyte, platelet, and lymphocyte counts. The SIRI and SII are used to predict the survival of patients with malignant tumors. It is well known that the inflammatory immune response is closely related to cancer occurrence and progression. In the present study, we evaluated the potential prognostic significance of SIRI and SII in patients with primary central nervous system lymphoma (PCNSL). METHODS: Fifty-eight consecutive patients were enrolled in this study between November 2006 and May 2022. Among the 58 patients, 47 patients with sufficient blood test data and follow-up were analyzed. The patients with steroid intake at the time point of the blood test and higher C-reactive protein were excluded. RESULTS: The median follow-up and survival times were 31 and 36 months, respectively. The optimal cutoff SIRI value was based on the receiver operating characteristic curve (ROC) for overall survival (OS) and stratified patients into low (< 1.43 × 109/L, n = 22) and high (≥ 1.43 × 109/L, n = 25) SIRI groups. The optimal cutoff SII value based on the ROC for OS stratified patients into low (< 694.9, n = 28) and high (≥ 694.9, n = 19) SII groups. A low SIRI value was associated with longer OS (p = 0.006). Furthermore, a low SII value was associated with longer OS (p = 0.044). The prognostic factors associated with prolonged survival in univariate analysis using the Cox proportional hazard model were age < 65 years, low SIRI, and low SII. The multivariate analysis demonstrated that age < 65 years and low SIRI independently predicted longer OS. CONCLUSION: Simple, less expensive, and routinely ordered preoperative blood count assessments such as SIRI and SII predict the OS of patients with PCNSL. This study demonstrated that PCNSL is associated with pre-treatment systemic immune-inflammation states.

Evaluating the Safety of Stent-Assisted Endovascular Treatment for Unruptured Cerebral Aneurysms in Older Adults: Emphasizing the Role of Antiplatelet Therapy.

INTRODUCTION: This study aimed to compare the outcomes and safety in patients aged ≥75 years and those aged <75 years who underwent stent-assisted endovascular treatment for unruptured cerebral aneurysms, specifically focusing on perioperative antiplatelet therapy (APT). METHODS: This multicenter retrospective study comprised patients who underwent stent-assisted coiling (SAC) or flow diverter stent (FDS) placement for unruptured cerebral aneurysms. The primary outcome was defined as the composite outcomes of perioperative thromboembolic events, bleeding events, or death. RESULTS: Among 632 patients, 533 (84.3%) were aged <75 years and 99 (15.6%) were aged ≥75 years. No significant differences were observed in the dual APT duration. The primary outcome occurred in 14.3% of patients aged <75 years and in 14.1% of those aged ≥75 years, with no significant difference (P=1.0). The composites of the primary outcome, including thromboembolic events, bleeding events, and death differed insignificantly. Similar findings were observed when the primary outcomes for SAC (12.7% vs. 11.5%, P=0.95) and FDS (17.5% vs. 18.4%, P=1.0) were analyzed. The 30-day, 1-year, and 2-year cumulative event-free survival rates for the primary outcome were 89.5, 87.2%, and 85.2%, respectively, in patients aged <75 years, and 90.9%, 88.7%, and 87.0%, respectively, in those aged ≥75 years. These trends were similar (log-rank test, P=0.92). CONCLUSION: No significant differences were observed in the rates of the primary outcomes between patients aged <75 years and those aged ≥75 years. Therefore, refraining from stent-assisted treatment for unruptured aneurysms based solely on age might be inappropriate.

Characterization of pig tonsils as niches for the generation of Streptococcus suis diversity.

Streptococcus suis is a gram-positive bacterium that causes meningitis, septicemia, endocarditis, and other disorders in pigs and humans. We obtained 42 and 50 S. suis isolates from lesions of porcine endocarditis and palatine tonsils, respectively, of clinically healthy pigs in Japan; we then determined their sequence types (STs) by multilocus sequence typing (MLST), cps genotypes, serotypes, and presence of classical major virulence-associated marker genes (mrp, epf, and sly). The 42 isolates from endocarditis lesions were assigned to a limited number of STs and clonal complexes (CCs). On the other hand, the 50 isolates from tonsils were diverse in these traits and seemingly in the degree of virulence, suggesting that tonsils can accommodate a variety of S. suis isolates. The goeBURST full algorithm using tonsil isolates obtained in this study and those retrieved from the database showed that major CCs as well as many other clusters were composed of isolates originating from different countries, and some of the STs were very similar to each other despite the difference in country of origin. These findings indicate that S. suis with not only different but also similar mutations in the genome have survived in tonsils independently across different geographical locations. Therefore, unlike the lesions of endocarditis, the tonsils of pigs seemingly accommodate various S. suis lineages. The present study suggests that S. suis acquired its diversity by natural mutations during colonization and persistence in the tonsils of pigs.

Medulla and cervical cord contrast-induced encephalopathy detected by neuromonitoring during coil embolization of posterior circulation aneurysms.

Understanding the risks of contrast-induced encephalopathy (CIE), a serious complication of contrast agents, is crucial in endovascular treatment. We present the case of a 73-year-old woman who developed CIE in the medulla and cervical cord during coil embolization for unruptured left basilar-superior cerebellar artery and basilar artery tip aneurysms. The CIE was identified via neuromonitoring. In this case, spinal cord ischemia might have occurred due to reduced perfusion pressure after inserting the distal access catheter (DAC) in the vertebral artery. Multiple injections of contrast medium via the DAC during coil embolization likely contributed to an unusual form of CIE. Extreme caution is warranted during endovascular treatments involving the posterior circulation, due to the relatively high incidence of contrast-mediated encephalopathy, which can lead to severe consequences such as perforator infarction. Neuromonitoring is very useful for the early detection of neurological changes, particularly because intraoperative angiography may not reveal all irregularities.

Intraoperative motor-evoked potential with tetanic stimulation changes pre- and post-hemispherotomy.

BACKGROUND: Careful examination of motor-evoked potential (MEP) findings is critical to the safety of intraoperative neuromonitoring during neurosurgery. We reviewed the intraoperative MEP findings in a pediatric patient who had undergone hemispherotomy for refractory epilepsy. CASE DESCRIPTION: The patient was a 4-year-and-2-month-old boy with extensive right cerebral hemisphere, drug-resistant epilepsy, left upper and lower extremity paralysis, and cognitive impairment. We examined intraoperative MEP results both before and after hemispherotomy. Post-hemispherotomy and MEPs were successfully elicited through transcranial electrical stimulation (TES) but not via direct cortical stimulation on the right side. Furthermore, TES on the right side, following hemispherotomy, led to a reduction in the MEP amplification effect resulting from tetanic stimulation of the left unilateral median and tibial nerves. Conversely, we observed the effects of MEP amplification during TES on the left side after tetanic stimulation of these nerves. Postoperatively, the patient underwent magnetic resonance imaging and electroencephalogram examinations, confirming the anatomical and electrophysiological completeness of the dissection. Notably, the seizures disappeared, and no apparent complications were observed. CONCLUSION: Collectively, our findings suggest that TES can still activate deep structures and elicit MEPs, even in cases where the corticospinal connections to the posterior limb of the internal capsule are entirely severed. Thalamo-cortical interactions may affect the MEP amplification, observed during tetanic stimulation. Injury to the corticospinal tracts of the white matter may be obscured on conventional MEP findings; however, it may be identified by MEP changes in tetanic stimulation.

Transcranial motor-evoked potentials disappear with pons transection.

Motor-evoked potential (MEP) monitoring by transcranial electrical stimulation (TES) is important for intraoperative motor function assessment in neurosurgery; however, false-negative results sometimes occur, and these findings should be interpreted with caution. Herein, we report an interesting MEP change resulting from a pons transection. The patient was a boy aged 5 years and 2 months. He underwent multiple craniotomies for cerebellar anaplastic ependymoma and was already paralyzed in the right upper and lower limbs. Therefore, we decided to remove the recurrent lesion from the left anterior pons. MEPs were recorded on both the right and left sides after the start of surgery but disappeared 1 h 30 min after the start of surgery in the TES on the operative side, even when the stimulation intensity was increased. The contralateral TES consistently recorded stable MEPs throughout the surgery. The tumor was completely resected on imaging. Immediately postoperatively, the patient experienced flaccid paralysis on the right side of the body, which recovered to preoperative levels over time. A transcranial MEP cannot be derived if the corticospinal tract is transected at the pons. Transcranial MEP findings may accurately reflect the corticospinal tract function if the injury is caudal to the pons.

Long-term outcomes and potential predictive recurrence factors after endonasal endoscopic surgical treatment of symptomatic Rathke's cleft cysts.

Although patients with symptomatic Rathke's cleft cysts (RCCs) receive surgical treatment, recurrence sometimes occurs after surgery. However, the mechanism underlying recurrence remains unclear. We evaluated the outcomes of RCC decompression over a long-term follow-up period. We retrospectively reviewed the medical records of 35 patients with symptomatic RCC who underwent endonasal endoscopic surgery (EES) at our institution between 2008 and 2023. Patients' characteristics, intraoperative findings, and postoperative follow-up outcomes were evaluated. A univariate regression model was used to identify the predictors of recurrence. The median patient age was 48.0 years, and 74.2% of the patients were female. The mean follow-up duration was 94.7 ± 47.6 months. Cyst content recurrence was observed in 15 patients (42.8%). Five patients (14.2%) with symptomatic recurrence underwent reoperation. Postoperative vision improved in all 23 patients (100%); headaches improved in 20 patients (90.9%). A new hormonal deficit occurred in two patients (5.7%). Complications included intraoperative cerebrospinal fluid (CSF) leak in 10 patients (28.5%), postoperative CSF leak in two patients (5.7%), permanent diabetes insipidus in two patients (5.7%), and postoperative infection in three patients (8.5%). Univariate analyses revealed that the position of the anterior pituitary lobe (p = 0.019) and preoperative visual disturbances (p = 0.008) significantly affected recurrence after surgery. Although EES was efficient, the recurrence rate was relatively high over a long-term period. The anterior pituitary lobe position and preoperative visual disturbances were significantly associated with recurrence. The anterior-inferior position can predict a high risk of recurrence before surgery.

Intraoperative monitoring of trigeminal neuralgia: a technical note.

Trigeminal neuralgia causes excruciating pain in patients. Microvascular decompression is indicated for drug-resistant s trigeminal neuralgia. Unlike facial spasms, any part of the nerve can be the culprit, not only the root entry zone. Intraoperative monitoring does not yet exist for trigeminal neuralgia. We successfully used intermittent stimulation of the superior cerebellar artery during surgery and confirmed the disappearance of the trigeminal nerve motor branch reaction after the release of the compression. Intermittent direct stimulation of the culprit blood vessel using the motor branch of the trigeminal nerve may assist in intraoperative monitoring of decompression during trigeminal nerve vascular decompression surgery.

Antitumor Effects of Intravenous Natural Killer Cell Infusion in an Orthotopic Glioblastoma Xenograft Murine Model and Gene Expression Profile Analysis.

Despite standard multimodality treatment, containing maximum safety resection, temozolomide, radiotherapy, and a tumor-treating field, patients with glioblastoma (GBM) present with a dismal prognosis. Natural killer cell (NKC)-based immunotherapy would play a critical role in GBM treatment. We have previously reported highly activated and ex vivo expanded NK cells derived from human peripheral blood, which exhibited anti-tumor effect against GBM cells. Here, we performed preclinical evaluation of the NK cells using an in vivo orthotopic xenograft model, the U87MG cell-derived brain tumor in NOD/Shi-scid, IL-2RɤKO (NOG) mouse. In the orthotopic xenograft model, the retro-orbital venous injection of NK cells prolonged overall survival of the NOG mouse, indirectly indicating the growth-inhibition effect of NK cells. In addition, we comprehensively summarized the differentially expressed genes, especially focusing on the expression of the NKC-activating receptors' ligands, inhibitory receptors' ligands, chemokines, and chemokine receptors, between murine brain tumor treated with NKCs and with no agents, by using microarray. Furthermore, we also performed differentially expressed gene analysis between an internal and external brain tumor in the orthotopic xenograft model. Our findings could provide pivotal information for the NK-cell-based immunotherapy for patients with GBM.

Characterization of HIF-1α Knockout Primary Human Natural Killer Cells Including Populations in Allogeneic Glioblastoma.

Enhancing immune cell functions in tumors remains a major challenge in cancer immunotherapy. Natural killer cells (NK) are major innate effector cells with broad cytotoxicity against tumors. Accordingly, NK cells are ideal candidates for cancer immunotherapy, including glioblastoma (GBM). Hypoxia is a common feature of solid tumors, and tumor cells and normal cells adapt to the tumor microenvironment by upregulating the transcription factor hypoxia-inducible factor (HIF)-1α, which can be detrimental to anti-tumor effector immune cell function, including that of NK cells. We knocked out HIF-1α in human primary NK cells using clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9). Then, cellular characterizations were conducted in normoxic and hypoxic conditions. Electroporating two HIF-1α-targeting guide RNA-Cas9 protein complexes inhibited HIF-1α expression in expanded NK cells. HIF-1α knockout human NK cells, including populations in hypoxic conditions, enhanced the growth inhibition of allogeneic GBM cells and induced apoptosis in GBM-cell-derived spheroids. RNA-sequencing revealed that the cytotoxicity of HIF-1α knockout NK cells could be related to increased perforin and TNF expression. The results demonstrated that HIF-1α knockout human NK cells, including populations, enhanced cytotoxicity in an environment mimicking the hypoxic conditions of GBM. CRISPR-Cas9-mediated HIF-1α knockout NK cells, including populations, could be a promising immunotherapeutic alternative in patients with GBM.

Clonal Expansion of Multidrug-Resistant Streptococcus dysgalactiae Subspecies equisimilis Causing Bacteremia, Japan, 2005-2021.

Incidence of Streptococcus dysgalactiae subspecies equisimilis (SDSE) bacteremia is increasing in the Kyoto-Shiga region of Japan. We retrospectively analyzed clinical features of SDSE bacteremia and conducted comparative genomic analyses of isolates collected from 146 bacteremia episodes among 133 patients during 2005-2021. Of those patients, 7.7% required vasopressor support, and 7.0% died while in the hospital. The prevalence of isolates resistant to erythromycin, minocycline, and clindamycin increased from 8.6% during 2005-2017 to 21.6% during 2018-2021. Our genomic analysis demonstrated that sequence type 525 and clonal complex 25 were predominant in SDSE isolates collected during 2018-2021. In addition, those isolates had acquired 2 antimicrobial-resistance genes, ermB and tetM, via Tn916-like integrative and conjugative elements (ICEs). Phylogenetic analysis revealed clonal distribution of Tn916-like ICEs in SDSE isolates. Our findings suggest that Tn916-like ICEs contributed to the emergence and recent increase of multidrug-resistant SDSE bacteremia in this region of Japan.

Group A Streptococcus cation diffusion facilitator proteins contribute to immune evasion by regulating intracellular metal concentrations.

Cation diffusion facilitators (CDFs) are a large family of divalent metal transporters with broad specificities that contribute to intracellular metal homeostasis and toxicity in bacterial pathogens. Streptococcus pyogenes (Group A Streptococcus [GAS]) expresses two homologous CDF efflux transporters, MntE and CzcD, which selectively transport Mn and Zn, respectively. We discovered that the MntE- and CzcD-deficient strains exhibited a marked decrease in the viability of macrophage-differentiated THP-1 cells and neutrophils. In addition, the viability of mice infected with both deficient strains markedly increased. Consistent with a previous study, our results suggest that MntE regulates the PerR-dependent oxidative stress response by maintaining intracellular Mn levels and contributing to the growth of GAS. The maturation and proteolytic activity of streptococcal cysteine protease (SpeB), an important virulence factor in GAS, has been reported to be abrogated by zinc and copper. Zn inhibited the maturation and proteolytic activity of SpeB in the culture supernatant of the CzcD-deficient strain. Furthermore, Mn inhibited SpeB maturation and proteolytic activity in a MntE-deficient strain. Since the host pathogenicity of the SpeB-deficient strain was significantly reduced, maintenance of intracellular manganese and zinc levels in the GAS via MntE and CzcD may not only confer metal resistance to the bacterium, but may also play an essential role in its virulence. These findings provide new insights into the molecular mechanisms of pathogenicity, which allow pathogens to survive under stressful conditions associated with elevated metal ion concentrations during host infection.

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

Embolic stroke induced by rotational persistent 1st intersegmental artery compression.

A 45-year-old man suffered multiple cerebral infarctions in the vertebrobasilar artery territory, followed by second stroke against conservative treatment. Radiological examinations revealed intra-arterial defect in left persistent 1st intersegmental artery (PFIA) at C1 level, suggesting mural thrombus, and mechanical compression of left PFIA at the level with head rotation to the right clearly revealed by reconstructed 3-dimensional radiological images, but no findings of atlantoaxial instability. One month after the second stroke, posterior fixation was performed. Postoperative course was uneventful without subsequent stroke for 24 months. This unique case demonstrated that PFIA might associate with cerebral stroke as a clinical condition of bow hunter's stroke even in middle age. Reconstructed 3-dimensional radiological images might be useful for clear demonstration of the pathophysiology in this complex clinical entity.

Natural Killer Cell-Based Immunotherapy against Glioblastoma.

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.

Therapeutic Anti-KIR Antibody of 1-7F9 Attenuates the Antitumor Effects of Expanded and Activated Human Primary Natural Killer Cells on In Vitro Glioblastoma-like Cells and Orthotopic Tumors Derived Therefrom.

Glioblastoma (GBM) is the leading malignant intracranial tumor, where prognosis for which has remained extremely poor for two decades. Immunotherapy has recently drawn attention as a cancer treatment, including for GBM. Natural killer (NK) cells are immune cells that attack cancer cells directly and produce antitumor immunity-related cytokines. The adoptive transfer of expanded and activated NK cells is expected to be a promising GBM immunotherapy. We previously established an efficient expansion method that produced highly purified, activated primary human NK cells, which we designated genuine induced NK cells (GiNKs). The GiNKs demonstrated antitumor effects in vitro and in vivo, which were less affected by blockade of the inhibitory checkpoint receptor programmed death 1 (PD-1). In the present study, we assessed the antitumor effects of GiNKs, both alone and combined with an antibody targeting killer Ig-like receptor 2DLs (KIR2DL1 and DL2/3, both inhibitory checkpoint receptors of NK cells) in vitro and in vivo with U87MG GBM-like cells and the T98G GBM cell line. Impedance-based real-time cell growth assays and apoptosis detection assays revealed that the GiNKs exhibited growth inhibitory effects on U87MG and T98G cells by inducing apoptosis. KIR2DL1 blockade attenuated the growth inhibition of the cell lines in vitro. The intracranial administration of GiNKs prolonged the overall survival of the U87MG-derived orthotopic xenograft brain tumor model. The KIR2DL1 blockade did not enhance the antitumor effects; rather, it attenuated it in the same manner as in the in vitro experiment. GiNK immunotherapy directly administered to the brain could be a promising immunotherapeutic alternative for patients with GBM. Furthermore, KIR2DL1 blockade appeared to require caution when used concomitantly with GiNKs.