Search

Particle dark matter could belong to a multiplet that includes an electrically charged state. WIMP dark matter (χ0) accompanied by a negatively charged excited state (χ−) with a small mass difference (e.g. < 20 MeV) can form a bound-state with a nucleus such as xenon. This bound-state formation is rare and the released energy is O(1−10) MeV depending on the nucleus, making large liquid scintillator detectors suitable for detection. We searched for bound-state formation events with xenon in two experimental phases of the KamLAND-Zen experiment, a xenon-doped liquid scintillator detector. No statistically significant events were observed. For a benchmark parameter set of WIMP mass mχ0=1 TeV and mass difference Δm=17 MeV, we set the most stringent upper limits on the recombination cross section times velocity 〈σv〉 and the decay-width of χ− to 9.2×10−30 cm3/s and 8.7×10−14 GeV, respectively at 90% confidence level.

Preceding a core-collapse supernova (CCSN), various processes produce an increasing amount of neutrinos of all flavors characterized by mounting energies from the interior of massive stars. Among them, the electron antineutrinos are potentially detectable by terrestrial neutrino experiments such as KamLAND and Super-Kamiokande (SK) via inverse beta decay interactions. Once these pre-supernova (pre-SN) neutrinos are observed, an early warning of the upcoming CCSN can be provided. In light of this, KamLAND and SK, both located in the Kamioka mine in Japan, have been monitoring pre-SN neutrinos since 2015 and 2021, respectively. Recently, we performed a joint study between KamLAND and SK on pre-SN neutrino detection. A pre-SN alert system combining the KamLAND detector and the SK detector was developed and put into operation, which can provide a supernova alert to the astrophysics community. Fully leveraging the complementary properties of these two detectors, the combined alert is expected to resolve a pre-SN neutrino signal from a 15 M _⊙ star within 510 pc of the Earth at a significance level corresponding to a false alarm rate of no more than 1 per century. For a Betelgeuse-like model with optimistic parameters, it can provide early warnings up to 12 hr in advance.

Immunotherapy improves survival outcomes in cancer patients, but there is still an unmet clinical need in the treatment of brain metastases. Here, we used a mouse model to investigate the antitumor effect of programmed death-ligand 1 (PD-L1) and vascular endothelial growth factor (VEGF) dual blockade on metastatic brain tumors and evaluated immune responses during treatment. After establishing hematogenous brain metastasis by transplanting murine bladder carcinoma MBT2 cells stably expressing secNLuc reporter via the internal carotid artery of C3H/HeNCrl mice, we observed the formation of metastases not only in the brain parenchyma but also in the ventricles. The observed pathological areas showed that metastases in the ventricle were histologically larger than that in the brain parenchyma. Regarding the total tumor burden in the whole brain as revealed by Nluc activities, the combination of anti-PD-L1 antibody and anti-VEGF antibody showed a stronger anti-tumor effect than each single agent. Anti-PD-L1 antibody alone enhanced CD8 + T cell priming in regional lymph nodes, increased the proportion of activated CD8 + T cells in whole brain, and increased the density of CD8 + cells in the brain parenchyma. Furthermore, anti-VEGF antibody alone decreased microvessel density (MVD) in ventricular metastases, and the combination treatment increased intratumoral CD8 + cell density in the brain parenchyma and ventricular metastases. These results suggest that PD-L1 blockade enhanced cancer immunity not only in brain metastases lesions but also in the regional lymph nodes of the metastases, and that the addition of VEGF blockade increased the antitumor effect by increasing the infiltration of activated CD8 + T cell and decreasing MVD., Competing Interests: Declarations Conflicts of interest Chinami Masuda, Shinichi Onishi, Keigo Yorozu, Mitsue Kurasawa, Mamiko Morinaga, Daiko Wakita, Masamichi Sugimoto are employees of Chugai Pharmaceutical Co., Ltd. Research involving human participants and/or animals All animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals at Chugai Pharmaceutical Co. Ltd., and all animal procedures were reviewed and approved by the Institutional Animal Care and Use Committee at Chugai Pharmaceutical Co., Ltd. Informed consent This article does not contain any studies involving human participants performed by any of the authors., (© 2024. The Author(s).)

Myasthenia gravis (MG) is a chronic autoimmune disease characterized by muscle weakness and fatigue. It is caused by pathological autoantibodies against components expressed at neuromuscular junctions, such as acetylcholine receptor (AChR). Interleukin-6 (IL-6) has been suggested to play a role in the pathogenesis of MG, and IL-6 receptor (IL-6R) antibody treatment may provide a novel therapeutic option. In this study, we investigated the effects of IL-6R antibody treatment in an experimental autoimmune MG (EAMG) mouse model. We demonstrated that IL-6R antibody treatment improved muscle weakness, reduced IgG deposition at neuromuscular junctions, and the levels of AChR autoantibodies in serum. In addition, follicular helper T cells and Th17, plasma cells in lymph nodes were lower in IL-6R antibody treated mice. Our findings suggest that IL-6R blockade may be a novel and effective therapeutic strategy for the treatment of MG., Competing Interests: SM, KS, SO, YK, MB, MK, HT-S, KY, YM, and MN-S were employed by Chugai Pharmaceutical Co., Ltd., (Copyright © 2024 Miyake, Serizawa, Onishi, Katsura, Baba, Kurasawa, Tomizawa-Shinohara, Yorozu, Matsumoto and Noguchi-Sasaki.)

Objective: An intact cochlear nerve is necessary for successful cochlear implantation (CI). Although the promontory stimulation test (PST) using a promontory stimulator (PS) and a transtympanic needle electrode is invasive, it is still commonly used to verify cochlear nerve function. PSs are currently unavailable because they are no longer manufactured; however, considering that PST continues to be beneficial in certain situations, alternative equipment is needed. The PNS-7000® (PNS) was developed as a neurologic instrument for stimulating the peripheral nerves. This study investigated the usefulness of the ear canal stimulation test (ECST) using PNS with a silver ball ear canal electrode, which is a new noninvasive alternative technique to the PST., Methods: ECST was performed from November 2013 to December 2018 using PS and PNS for patients with severe to profound sensorineural hearing loss. The electrical threshold, most comfortable loudness level, uncomfortable loudness level, dynamic range, and gap detection were measured in the ECST. The results of these measured PNS items were compared with PS., Results: ECST was performed in 61 ears of 35 patients (age, 59.9 ± 20.1 years) using PS and PNS. The sound sensation was elicited in 51 (83.6%) and 52 (85.2%) ears with PS and PNS, respectively. All items excluding GAP were measured in 46 (75%) and 43 (70%) ears at 50 and 100 Hz, respectively. GAP was measured in 33 ears by the ascending and descending methods using PS and PNS. Spearman's rank-order correlation coefficient revealed a significant positive linear correlation between the PS and PNS results in all measurements. No significant difference was found between the PS and PNS thresholds in all measured items., Conclusions: PNS is a useful instrument for performing ECST as a new alternative to PS. ECST using a silver ball electrode is a less invasive and easier test than PST., Competing Interests: Declaration of Competing Interest The authors have no financial conflicts of interest to declare., (Copyright © 2023. Published by Elsevier B.V.)

Several reports assume that myocardial necroptotic cell death is induced during the development of chronic heart failure. Although it is well accepted that angiotensin II induces apoptotic cell death of cardiac myocytes, the involvement of angiotensin II in the induction of myocardial necroptosis during the development of heart failure is still unknown. Therefore, we examined the role of angiotensin II in myocardial necroptosis using rat failing hearts following myocardial infarction and cultured cardiomyocytes. We found that administration of azilsartan, an angiotensin II AT 1 receptor blocker, or trandolapril, an angiotensin-converting enzyme inhibitor, to rats from the 2nd to the 8th week after myocardial infarction resulted in preservation of cardiac function and attenuation of mixed lineage kinase domain-like (MLKL) activation. Furthermore, the ratio of necroptotic cell death was increased in neonatal rat ventricular cardiomyocytes cultured with conditioned medium from rat cardiac fibroblasts in the presence of angiotensin II. This increase in necroptotic cells was attenuated by pretreatment with azilsartan. Furthermore, activated MLKL was increased in cardiomyocytes cultured in conditioned medium. Pretreatment with azilsartan also prevented the conditioned medium-induced increase in activated MLKL. These results suggest that angiotensin II contributes to the induction of myocardial necroptosis during the development of heart failure.

We challenged to create a mouse model of neuromyelitis optica spectrum disorder (NMOSD) induced by AQP4 peptide immunization. Intradermal immunization with AQP4 p201-220 peptide induced paralysis in C57BL/6J mice, but not in AQP4 KO mice. AQP4 peptide-immunized mice showed pathological features similar to NMOSD. Administration of anti-IL-6 receptor antibody (MR16-1) inhibited the induction of clinical signs and prevented the loss of GFAP/AQP4 and deposition of complement factors in AQP4 peptide-immunized mice. This novel experimental model may contribute to further understanding the pathogenesis of NMOSD, elucidating the mechanism of action of therapeutic agents, and developing new therapeutic approaches., Competing Interests: Declaration of Competing Interest All authors are employees of Chugai Pharmaceutical Co., Ltd., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The KamLAND-Zen experiment has provided stringent constraints on the neutrinoless double-beta (0νββ) decay half-life in ^{136}Xe using a xenon-loaded liquid scintillator. We report an improved search using an upgraded detector with almost double the amount of xenon and an ultralow radioactivity container, corresponding to an exposure of 970 kg yr of ^{136}Xe. These new data provide valuable insight into backgrounds, especially from cosmic muon spallation of xenon, and have required the use of novel background rejection techniques. We obtain a lower limit for the 0νββ decay half-life of T&#95;{1/2}^{0ν}>2.3×10^{26}  yr at 90% C.L., corresponding to upper limits on the effective Majorana neutrino mass of 36-156 meV using commonly adopted nuclear matrix element calculations.

Gene amplification and protein overexpression of human epidermal growth factor receptor type 2 (HER2) are specific targets for HER2-targeting drugs in breast, gastric, salivary gland, and colorectal cancers. The histopathological determination of HER2 status is crucial for treatment, highlighting the importance of improving HER2 detection accuracy in clinical practice. We prepared tissue microarray (TMA) slides for use as control slides for the standardization of gastric HER2 testing. Four human gastric cancer cell lines with HER2 scores of 3+, 2+, 1+, and 0 were xenografted in NOG mice. The TMA slides were constructed using samples from three different areas in these tumors. Staining properties were determined using six clinical kits for HER2. In TMA, HER2-positive tumors with HER2 scores of 3+ and 2+ showed good staining with all diagnostic kits, and the tissue images were similar to those of clinical samples. Xenograft tumor slides could potentially be used as external controls to standardize staining conditions for a variety of kits and may improve the accuracy of HER2 detection in clinical practice., (© 2022 The Authors. Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Objectives: This study aimed to quantitatively assess maxillary central incisor roots using pre-orthodontics computed tomography (CT) texture analysis as part of a radiomics quantitative analysis., Methods: This retrospective case-control study included 16 patients with external apical root resorption (EARR) and 16 age- and sex-matched patients without EARR, after orthodontic treatment who underwent pre-orthodontics CT for jaw deformities. All patients were treated with a fixed orthodontic appliance before and after surgical orthodontic treatment. EARR was defined as root resorption ≥ 2 mm of the left and right maxillary central incisors on CT images more than 2 years after the start of orthodontic treatment. Texture features of the maxillary central incisor with and without EARR after orthodontic treatment were analyzed using the open-access software, MaZda Ver. 3.3. Ten texture features were selected using the Fisher method in MaZda from 279 original parameters, which were calculated for each of the maxillary central incisors with and without EARR. The results were tested using the Student's t test, Welch's t test, or Mann-Whitney U test., Results: Four gray-level run length matrix features and six gray-level co-occurrence matrix features displayed significant differences between both the groups (p < 0.01)., Conclusions: CT texture analysis was able to quantitatively assess maxillary central incisor roots and distinguish between maxillary central incisor roots with and without EARR. CT texture analysis may be a useful method for predicting EARR after orthodontic treatment., (© 2022. The Author(s) under exclusive licence to Japanese Society for Oral and Maxillofacial Radiology.)

Background/aim: Although CDK4/6 inhibitors have been increasingly used in combination with hormonal agents to treat hormone-receptor positive and human epithelial growth factor receptor 2-negative breast cancer, the mechanism of CDK4/6 inhibitor resistance and its impact on established therapy for post-resistance, especially bevacizumab combined with chemotherapy, are unclear., Materials and Methods: Sensitivity of RB knockout MCF7 clones to CDK4/6 inhibitors was evaluated in vitro. One RB knockout clone was subcutaneously implanted in nude mice and the effects of bevacizumab on volume and microvessel density (MVD) of tumors were investigated., Results: Palbociclib did not exhibit antitumor efficacy against the RB knockout tumor, in contrast to the parental MCF7 xenograft model. Bevacizumab significantly exhibited antitumor efficacy and suppressed the MVD both in RB knockout and parental MCF7 xenograft models., Conclusion: Bevacizumab inhibited tumor growth by suppressing MVD in the CDK4/6 inhibitor-resistant tumor acquired due to RB loss, suggesting its efficacy also in patients after treatment with CDK4/6 inhibitors., Competing Interests: All the Authors are employees of Chugai Pharmaceutical Co., Ltd., Tokyo, Japan., (Copyright 2022, International Institute of Anticancer Research.)

Background: The glycoengineered, humanized anti-CD20 antibody obinutuzumab is indicated for previously untreated or relapsed/refractory CD20-positive follicular lymphoma (FL). However, the effectiveness of obinutuzumab retreatment in relapsed/refractory FL after prior obinutuzumab-containing therapy is unclear. To address this issue, we investigated the antitumor activity of obinutuzumab plus bendamustine in obinutuzumab-resistant tumors established from a human non-Hodgkin lymphoma xenograft model., Materials and Methods: Obinutuzumab-resistant tumors (SU-DHL-4-OR-18-8) were established from an SU-DHL-4 xenograft model by repeated administration of obinutuzumab. Antitumor activity was evaluated based on tumor volume after treatment with obinutuzumab on Day 1, 8, and 15 and/or bendamustine on Day 1 and 2. Intratumoral natural killer (NK) cells/macrophages were evaluated by immunohistochemistry and flow cytometry., Results: In SU-DHL-4-OR-18-8 xenografted tumors, intratumoral NK cells/macrophages after obinutuzumab treatment were significantly decreased compared with parent tumors on Day 4. The endoplasmic reticulum stress sensor phospho-IRE1 was also decreased. In SU-DHL-4-OR-18-8 tumors, bendamustine treatment increased phospho-IRE1 on Day 4 and intratumor NK cells/macrophages on Day 10. Obinutuzumab combined with bendamustine significantly increased antitumor activity compared with each single agent on Day 29, with an increase in chemoattractant CCL6 expression on Day 10., Conclusions: Coadministration of bendamustine in obinutuzumab retreatment may be effective against obinutuzumab-resistant tumors., (© 2022. Japanese Society of Hematology.)

Brain metastases are common complication in cancer patients. Immune checkpoint inhibitors show therapeutic benefits also in patients with central nervous system (CNS) metastases. However, their antitumor effects on metastatic tumors and their underlying mechanisms are still poorly understood. In this study we investigated the antitumor effect of anti-programmed death-ligand 1 (PD-L1) antibody on metastatic brain tumors and evaluated immune responses during treatment. We employed a hematogenous brain metastasis xenograft model using immunodeficient mice with murine lymphocyte infusions. A human non-small-cell lung cancer (NSCLC) cell line stably expressing NanoLuc ® reporter (Nluc-H1915) was inoculated from the internal carotid artery of SCID mice. After metastases were established (24 days after inoculation), splenocytes prepared from H1915-immunized BALB/c mice were injected intravenously and mouse IgG or anti-PD-L1 antibody treatment was started (day 1). Evaluated by Nluc activity, tumor volume in the brain on day 14 was significantly lower in anti-PD-L1-treated mice than in mouse IgG-treated mice. Furthermore CD8 + cells were primarily infiltrated intratumorally and peritumorally and anti-PD-L1 treatment induced a significantly higher proportion of Granzyme B (GzmB) + cells among CD8 + T cells. The antitumor effect of anti-PD-L1 antibody on brain metastasis is thought to be achieved by the enhanced activation of infiltrated CD8 + T cells into metastatic brain tumor. These results suggest that anti-PD-L1 antibody-containing regimens may be promising treatment options for cancer patients with brain metastases., (© 2021. The Author(s).)

The efficacy of programmed cell death‑ligand 1 (PD‑L1)/programmed cell death protein 1 (PD‑1) blockade therapy has been demonstrated but is limited in patients with PD‑L1 low or immune desert tumors. This limitation can be overcome by combination therapies that include anti‑vascular endothelial growth factor (VEGF) therapy. Such combinations have been investigated in clinical trials for a number of cancer types; however, evidence on the mechanisms underlying their effects in these types of patients is still not sufficient. Therefore, the present study investigated the efficacy and effects on CD8 + T cell and C‑X‑C motif chemokine receptor 3 (CXCR3) ligand expression in tumors by combining anti‑PD‑L1 and anti‑VEGF antibodies using an OV2944‑HM‑1 mouse model with PD‑L1 low and immune desert‑like phenotypes. Although the model exhibited anti‑PD‑L1 insensitivity, anti‑PD‑L1 antibody treatment combined with anti‑VEGF antibody inhibited tumor growth compared with anti‑VEGF monotherapy, which itself inhibited tumor growth compared with the control treatment on Day 25. In combination‑treated mice, a higher percentage of CD8 + T cells and higher levels of CXCR3 ligands were observed in tumor tissues compared with those in the anti‑VEGF antibody treatment group, which was not significantly different from control treatment on Day 8. The increase in the intratumoral percentage of CD8 + T cells following the combination treatment was reversed by CXCR3 blocking to the same level as the control. In an anti‑PD‑L1 insensitive model with PD‑L1 low and immune desert‑like phenotypes, although anti‑PD‑L1 antibody alone was not effective, anti‑PD‑L1 antibody in combination with anti‑VEGF antibody exhibited antitumor combination efficacy with an increase of CD8 + T cell infiltration, which was suggested to be dependent on the increase of intratumoral CXCR3 ligands. This mechanism could explain the efficacy of anti‑PD‑L1 antibody and anti‑VEGF antibody combination therapy in the clinical setting.

Immune-related pneumonitis is an important toxicity associated with checkpoint inhibitor therapy with anti-PD-1 or anti-PD-L1 antibodies, often necessitating discontinuation of treatment. Development of methods to mitigate checkpoint inhibitor-related pneumonitis is required.The contributions of PD-L1, PD-L2, and VEGF to the pathogenesis of pneumonitis were examined in an IL2- plus IL18-induced mouse pneumonitis model (IL pneumonitis model). Furthermore, the incidences of pneumonitis were retrospectively examined in patients with non-small cell lung cancer treated with the anti-PD-L1 mAb atezolizumab plus chemotherapy, with or without the anti-VEGF mAb bevacizumab, in the phase III IMpower150 trial. PD-1 signal blockade by anti-PD-L1 and anti-PD-L2 antibodies aggravated pneumonitis in the IL pneumonitis model. An anti-VEGF antibody prevented PD-1 signal blockade from aggravating pneumonitis in this model. PD-1 signal blockade induced interstitial T-cell infiltration in the lungs, but VEGF blockade did not affect this T-cell infiltration. The anti-VEGF antibody protected against vascular-to-alveolar leakage of protein and fluid due to PD-1 signal blockade in a murine model. In the IMpower150 trial, incidence rates of pneumonitis of any grade were 4.3% in the group without bevacizumab and 2.8% in the group with bevacizumab. In patients with pneumonitis, outcomes of "Not recovered/Not resolved" were reported for 29.4% in the group without bevacizumab compared with 9.1% in the group with bevacizumab. Our findings suggest that anti-VEGF antibodies in combination with checkpoint inhibitors may be a treatment method that can control checkpoint inhibitor-related pneumonitis., (©2021 The Authors; Published by the American Association for Cancer Research.)

Anti-PD-L1 antibodies benefit many cancer patients, even those with "non-inflamed tumor". Determining which patients will benefit remains an important clinical goal. In a non-inflamed tumor mouse model, we found that PD-L1 was highly expressed on antigen-presenting cells (APCs) especially on CD103 + CD11c + dendritic cells in tumor-draining lymph nodes (dLNs), suppressing T-cell priming by APCs. In this model, anti-PD-L1 antibodies enhanced T-cell priming and increased CXCR3 + activated T-cells in dLNs, which was followed by the trafficking of T-cells to tumors in response to CXCR3 ligands. As predictive biomarker, each APCs-related gene expression (AP score) alone or T-cells trafficking-related chemokine gene expression (T score) alone were still less than perfect among the 17 mouse models examined. However a combining score of AP score and T score (AP/T score) precisely identified anti-PD-L1-sensitive tumors. In the phase 3 trial of atezolizumab vs docetaxel in advanced NSCLC patients (OAK), the AP/T score could identify atezolizumab-treated NSCLC patients who achieved significant improvement in overall survival. This biomarker concept would be a clinically valuable for prediction of anti-PD-L1 antibody efficacy.

Purpose: Complex fetal behavior involving multiple parts of the body, called general movement (GM), has been considered an essential predictor of neurological functional development because it directly reflects the integrity of the brain and central and peripheral nervous systems. We have developed a novel method for quantitative analysis of fetal behavior using four-dimensional ultrasound (4DUS) and conducted a pilot study for quantitative assessment of fetal GM in the early second trimester., Methods: All subjects underwent 4DUS to depict the whole fetal body, and maximum velocity (MAXV), median velocity (MV), average velocity (AV), and mode velocity (MOV) were calculated by utilizing optical flow analysis. Receiver operating characteristic (ROC) curve analysis was performed to analyze the optimal speed parameters for detecting GM in the fetus. The Mann-Whitney U test was used to validate MAXV, AV, and MV ability to detect fetal GM., Results: The presence of fetal GMs and the absence of fetal GMs were 226 and 107, respectively, based on optical flow analysis. Mann-Whitney U test revealed a significant difference in the presence or absence of fetal GM in MAXV, MV, AV, and MOV. ROC analysis showed that the area under the curve (AUC) of MAXV was 0.959; the threshold was 0.421, the sensitivity was 86%, and the specificity was 93%. In contrast, the AUC/threshold for AV and MV was 0.700/0.110 (sensitivity 71% and specificity 76%) and 0.521/0.119 (sensitivity 21% and specificity 90%), respectively. Spearman's rank correlation analysis also showed a weak negative correlation between GM and MAXV (r = - 0.235, P < 0.01) and AV (r = - 0.28, P < 0.01)., Conclusion: In this study, we conducted a quantitative analysis of fetal behavior based on optical flow using 4DUS and demonstrated that it was highly accurate for detecting GMs and for evaluating developmental changes in GMs. The implementation of quantitative analysis of fetal GMs in the early second trimester has been very preliminary, and there is much debate on how it will be clinically applied to perinatal assessment., (© 2021. The Japan Society of Ultrasonics in Medicine.)

Brain metastases are common in patients with non-small-cell lung cancer (NSCLC). The efficacy of bevacizumab, an anti-vascular endothelial growth factor (VEGF) humanized antibody, has been demonstrated in patients with nonsquamous NSCLC. We established a transplantable NSCLC cell line (Nluc-H1915) that stably expresses NanoLuc® reporter and confirmed the correlation between total Nluc activity in tumor and tumor volume in vivo. SCID mice inoculated with these cells through the internal carotid artery formed reproducible brain metastases, in which human VEGF was detected. Next, after metastases were established in the model mice (15-17 days), they were intraperitoneally administered weekly doses of human immunoglobulin G (HuIgG) or bevacizumab. Nluc activity in the brain was significantly lower in bevacizumab-treated mice than in HuIgG-treated mice. Additionally, bevacizumab concentration in the brain was higher in mice with brain metastasis than in normal mice, and bevacizumab was primarily observed in brain metastasis lesions. The microvessel density in brain metastasis was lower in bevacizumab-treated mice than in HuIgG-treated mice. We believe bevacizumab's anti-proliferative effect on brain metastasis is due to anti-angiogenic activity achieved by its penetration into brain metastases; this suggests that a bevacizumab-containing regimen may be a promising treatment option for patients with NSCLC brain metastasis.

Aim: Iron overload is a life-threatening disorder that can increase the risks of cancer, cardiovascular disease, and liver cirrhosis. There is also a risk of iron overload in patients with chronic kidney disease. In patients with renal failure, iron storage is increased due to inadequate iron utilization associated with decreased erythropoiesis and also to the inflammatory status. To evade the risk of iron overload, an accurate and versatile indicator of body iron storage in patients with iron overload is needed. In this study, we aimed to find useful iron-related parameters that could accurately reflect body iron storage in mice in order to construct a murine model of iron overload., Methods: To select an appropriate indicator of body iron status, a variety of parameters involved in iron metabolism were evaluated. Noninvasively measured parameters were R1, R2, and R2 ∗ derived from magnetic resonance imaging (MRI). Invasively measured parameters included serum hepcidin levels, serum ferritin levels, and liver iron contents. Histopathological analysis was also conducted., Results/conclusion: Among the several parameters evaluated, the MRI T2 ∗ relaxation time was able to detect iron storage in the liver as sensitively as serum ferritin levels. Moreover, it is expected that using an MRI parameter will allow accurate evaluation of body iron storage in mice over time., Competing Interests: The authors declare that they have no competing interests., (Copyright © 2019 Yukari Matsuo-Tezuka et al.)

Although bevacizumab maintenance following bevacizumab in combination with chemotherapy has demonstrated significant prolongation of progression-free survival in clinical studies in patients with ovarian cancer, the majority of the cancer cases in the study were of the serous histotype; therefore, data regarding clear cell carcinoma is limited. Furthermore, the efficacy of bevacizumab beyond progression has not yet been demonstrated in ovarian cancer. A xenograft model using the human ovarian clear cell carcinoma cell line RMG-I was used to investigate the antitumor effects and the mechanisms of bevacizumab in maintenance treatment and bevacizumab when administered beyond disease progression. In the RMG-I model, bevacizumab maintenance following bevacizumab in combination with paclitaxel exhibited increased tumor suppression, compared with its absence, and inhibited the increase of microvessel density (MVD) in tumors. Following disease progression during bevacizumab maintenance, continued bevacizumab treatment in combination with PEGylated liposomal doxorubicin as a secondary chemotherapeutic agent had increased efficacy, compared with PEGylated liposomal doxorubicin alone, and resulted in lower MVD accompanied with lower levels of insulin-like growth factor binding protein-3, which is reported to have angiogenic activity. Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of bevacizumab maintenance and bevacizumab beyond progression in ovarian cancer.

Anti-PD-L1 antibodies inhibit interactions between PD-L1 and PD-1 and interactions between PD-L1 and B7-1, thereby reinvigorating anticancer immunity. Although there are numerous ongoing clinical studies evaluating combinations of standard chemotherapies and anti-PD-L1 antibodies, irinotecan has not yet been investigated in this context so there is little information about its compatibility with anti-PD-L1 antibodies. Here we investigated the efficacy of anti-PD-L1 antibody in combination with irinotecan and the role of irinotecan in the tumor-immunity cycle in an FM3A murine tumor model. Despite a transient decrease in lymphocytes in the peripheral blood after irinotecan treatment, the antitumor activity of anti-PD-L1 antibody plus irinotecan was significantly greater than each agent alone. Irinotecan in combination with anti-PD-L1 antibody enhanced proliferation of CD8 + cells in both tumors and lymph nodes, and the number of tumor-infiltrating CD8 + cells was higher than either irinotecan or anti-PD-L1 antibody monotherapy. Irinotecan was found to decrease the number of Tregs in lymph nodes and tumors, and specific depletion of Tregs by anti-folate receptor 4 antibodies was found to enhance the proliferation of CD8 + cells in this model. In addition, irinotecan augmented MHC class I expression on tumor cells and concurrently increased PD-L1 expression on tumor cells and tumor-infiltrating immune cells. These results indicate that irinotecan may enhance the effect of T cell activation caused by anti-PD-L1 treatment by reducing Tregs and augmenting MHC class I-mediated tumor antigen presentation, and concurrent upregulation of PD-L1 expression can be blocked by the anti-PD-L1 antibody. These interactions may contribute to the superior combination effect., Competing Interests: CONFLICTS OF INTEREST All authors are employees of Chugai Pharmaceutical Co., Ltd.

Epoetin beta pegol (continuous erythropoiesis receptor activator; C.E.R.A.), or methoxy-polyethylene glycol-modified epoetin beta, is a long-acting erythropoiesis stimulating agent (ESA) that effectively maintains hemoglobin levels. It promotes proliferation of erythroid progenitor cells in hematopoietic organs and leads to increased reticulocyte and hemoglobin levels. However, the detailed erythropoietic effects of various ESAs on their target organs have yet to be clarified, and new approaches are needed to analyze tissue iron localization with structural information. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) techniques are widely used in basic pharmaceutical research. High-resolution Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS) imaging enables the spatial mapping and identification of biomolecules. In this study, mice administered with C.E.R.A. were fed a diet containing the stable iron isotope 57 Fe. The 57 Fe-heme + isotopic fine structure peak (m/z 617.1772) was separated from the non-labeled heme + isotopic peak (Δ0.0029) by FTICR-MS with a resolving power of more than 500,000. We optimized the platform to analyze the distribution of 57 Fe-heme in the spleen using MALDI FTICR-MS imaging. The combination of the ultrahigh resolution power of FTICR-MS and a stable isotope labeling technique has the potential to be very effective in basic pharmaceutical research. Graphical Abstract ᅟ.

Erlotinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), shows superior efficacy in patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations (EGFR Mut+). However, almost all tumors eventually develop resistance to erlotinib. Recently, the Phase II JO25567 study reported significant prolongation of progression-free survival (PFS) by erlotinib plus bevacizumab combination compared with erlotinib in EGFR Mut+ NSCLC. Herein, we established a preclinical model which became refractory to erlotinib after long-term administration and elucidated the mode of action of this combination. In this model, tumor regrowth occurred after remarkable shrinkage by erlotinib; regrowth was successfully inhibited by erlotinib plus bevacizumab. Tumor vascular endothelial growth factor (VEGF) was greatly reduced by erlotinib in the erlotinib-sensitive phase but significantly increased in the erlotinib-refractory phase despite continued treatment with erlotinib. Although EGFR phosphorylation remained suppressed in the erlotinib-refractory phase, phosphorylated extracellular signal-regulated kinase (pERK), phosphorylated AKT, and phosphorylated signal transducer and activator of transcription 3 (pSTAT3) were markedly higher than in the erlotinib-sensitive phase; among these, pERK was suppressed by erlotinib plus bevacizumab. MVD was decreased significantly more with erlotinib plus bevacizumab than with each drug alone. In conclusion, the erlotinib plus bevacizumab combination demonstrated promising efficacy in the B901L xenograft model of EGFR Mut+ NSCLC. Re-induction of VEGF and subsequent direct or indirect VEGF-dependent tumor growth was suggested as a major mechanism of erlotinib resistance, and erlotinib plus bevacizumab achieved remarkably prolonged antitumor activity in this model.

Erythropoiesis-stimulating agents are among the therapeutic options for renal anemia. Under erythropoietic stimulation, the synthesis of hemoglobin requires a large amount of iron, which is supplied both from absorption of dietary iron and the mobilization of stored iron. However, under iron-loading conditions, dietary iron absorption is suppressed via down-regulation of duodenal iron transporters. Because the contribution of dietary iron is essential for erythropoiesis, we aimed to investigate the conditions in which dietary iron is efficiently used for erythropoiesis. To quantify the contribution of dietary iron for erythropoiesis, we labelled dietary iron using the stable iron isotope 57 Fe, and assessed 57 Fe-derived hemoglobin synthesis under erythropoietic stimulation by C.E.R.A. (Continuous Erythropoietin Receptor Activator) and iron loading in mice. Our results show that the contribution of dietary iron to erythropoiesis is not changed by different increments of C.E.R.A. dose. However, iron loading suppressed the contribution of dietary iron to erythropoiesis. To confirm these results under conditions mimicking clinical settings, we compared single and intermittent administration of C.E.R.A. in mice under both physiological and iron-loaded conditions, and found that the contribution of dietary iron to erythropoiesis is more strongly affected by body iron status than by erythropoietic stimulation.

Background: Bevacizumab in combination with chemotherapeutics has shown significant survival benefit in clinical studies in patients with non-small cell lung cancer (NSCLC). Since bevacizumab was administered as standard treatment until disease progression, the importance of bevacizumab during the maintenance phase was not prospectively investigated in these studies., Materials and Methods: Three human NSCLC cell line xenograft models were used to investigate antitumor effect of bevacizumab and tumor microvessel density (MVD) was analyzed., Results: In A549 and NCI-H292 models, bevacizumab maintenance following combination with paclitaxel exhibited stronger tumor suppression than its absence. In an NCI-H292 model, bevacizumab maintenance continuously inhibited increase of MVD. In an NCI-H2228 model following induction treatment with pemetrexed and bevacizumab, maintenance with pemetrexed plus bevacizumab, had stronger efficacy than pemetrexed alone and led to lower MVD and level of thymidylate synthase., Conclusion: Continuous suppression of angiogenesis by bevacizumab may contribute to the superior efficacy of maintenance treatment containing bevacizumab., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Vascular endothelial growth factor (VEGF)-neutralizing therapy with bevacizumab has become increasingly important for treating colorectal cancer. It was demonstrated that second-line chemotherapy together with bevacizumab after disease progression (PD) on first-line therapy including bevacizumab showed clinical benefits in metastatic colorectal and breast cancers (ML18147 trial, TANIA trial). One of the rationales for these trials was that the refractoriness to first-line therapy is caused by resistance to not so much bevacizumab as to the chemotherapeutic agents. Nevertheless, resistance to bevacizumab cannot be ruled out because VEGF-independent angiogenesis has been reported to be a mechanism of resistance to anti-VEGF therapy. In this study, we used a xenograft model with the human colon cancer HT-29 cells to investigate the mechanisms underlying the effect of continued administration of bevacizumab plus capecitabine even after resistance to bevacizumab was acquired. The combination of capecitabine plus bevacizumab exhibited significantly stronger antitumor and anti-angiogenic activities than did monotherapy with either agent. Capecitabine treatment significantly increased the intratumoral VEGF level compared with the control group; however, the combination with bevacizumab neutralized the VEGF. Among angiogenic factors other than VEGF, intratumoral galectin-3, which reportedly promotes angiogenesis both dependent on, and independently of VEGF, was significantly decreased in the capecitabine group and the combination group compared with the control group. In an in vitro experiment, 5-fluorouracil (5-FU), an active metabolite of capecitabine, inhibited galectin-3 production by HT-29 cells. These results suggested that capecitabine has a dual mode of action: namely, inhibition of tumor cell growth and inhibition of galectin-3 production by tumor cells. Thus, capecitabine and bevacizumab may work in a mutually complementary manner in tumor angiogenesis inhibition to overcome the resistance caused by angiogenic factors other than VEGF. These results suggest the clinical relevance and the mechanism of action of treatment with bevacizumab in combination therapy beyond PD.

Erythropoiesis requires large amounts of iron for hemoglobin synthesis. There are two sources of iron for erythropoiesis, dietary and stored iron; however, their relative contributions to erythropoiesis remain unknown. In this study, we used the stable iron isotope (57)Fe to quantify synthesis of hemoglobin derived from dietary iron. Using this method, we investigated the activities of dietary iron absorption and the utilization of dietary iron for erythropoiesis in responses to stimulated erythropoiesis and to interventions to alter body iron status. Under iron-loaded conditions, the activity of dietary iron absorption was clearly lowered in response to up-regulation of hepcidin, although the estimated activity of iron release from stored iron was not compared with that under control conditions. This result was supported by the observation that two duodenal iron transporters, divalent metal transporter 1 (DMT1) and ferroportin, were downregulated by iron loading, although the levels of expression of ferroportin in iron storage tissues were not changed by iron loading under erythropoietic stimulation by epoetin-β pegol (C.E.R.A., a long-acting erythropoiesis-stimulating agent). These results indicate that the dietary iron absorption system is more sensitive to body iron status than are reticuloendothelial iron- release mechanisms. Our data indicated that there could be a regulatory mechanism favoring use of stored iron over dietary iron under iron-loaded conditions., (Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.)

Unlabelled: Iron, an essential element for various biological processes, can induce oxidative stress. We hypothesized that iron utilization for erythropoiesis, stimulated by epoetin beta pegol (C.E.R.A.), a long-acting erythropoiesis-stimulating agent, contributes to the reduction of iron-induced oxidative stress. We first investigated the sensitivity of several biomarkers to detect oxidative stress in mice by altering the amount of total body iron; we then investigated whether C.E.R.A. ameliorated oxidative stress through enhanced iron utilization. We treated db/db mice with intravenous iron-dextran and evaluated several biomarkers of iron-induced oxidative stress. In mice loaded with 5 mg/head iron, hepatic iron content was elevated and the oxidative stress marker d-ROMs (serum derivatives of reactive oxygen metabolites) was increased, whereas urinary 8-hydroxy-2'-deoxyguanosine and serum malondialdehyde were not, indicating that d-ROMs is a sensitive marker of iron-induced oxidative stress. To investigate whether C.E.R.A. ameliorated oxidative stress, db/db mice were intravenously administered iron-dextran or dextran only, followed by C.E.R.A. Hemoglobin level increased, while hepatic iron content decreased after C.E.R.A., Treatment: Serum d-ROMs decreased after C.E.R.A. treatment in the iron-dextran-treated group. Our results suggest that C.E.R.A. promotes iron utilization for erythropoiesis through mobilization of hepatic iron storage, leading to a decrease in serum oxidative stress markers in iron-loaded db/db mice.

Purpose: Alectinib is a highly selective next-generation anaplastic lymphoma kinase (ALK) inhibitor. Although alectinib shows inhibitory activity against various crizotinib-resistant ALK mutations in studies using cell-free kinase assays and Ba/F3 cell-based assays, it has not been tested for efficacy against non-small cell lung cancer (NSCLC) with the ALK mutations., Methods: We conducted in vitro and in vivo investigations into the antitumor activity of alectinib against an ALK-positive NSCLC cell line, SNU-2535, which harbors an ALK G1269A mutation. The clinical efficacy of alectinib against a NSCLC patient harboring ALK G1269A mutation was evaluated in the phase I part of the North American study., Results: Alectinib exhibited antiproliferative activity against SNU-2535 cells in vitro with IC50 of 33.1 nM. Alectinib strongly inhibited phosphorylation of ALK and its downstream signaling molecules ERK1/2, AKT, and STAT3. In a mouse xenograft model, once-daily oral administration of alectinib for 21 days resulted in strong tumor regression. In addition, administration of alectinib for 100 days achieved continuous tumor regression without tumor regrowth in all mice. Notably, eradication of tumor cells was observed in half of the mice. In the clinical study, a patient with ALK G1269A mutation showed partial response to alectinib with a duration of response of 84 days., Conclusion: These results indicated that alectinib has potent antitumor activity against NSCLC cells harboring the crizotinib-resistant mutation ALK G1269A. It is expected that alectinib would provide a valuable therapeutic option for patients with NSCLC having not only native ALK but also crizotinib-resistant ALK mutations.

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non-small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression-free survival in patients with EGFR-mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR-mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR-mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug-cessation period. In the HCC827-EPR model (DEL+T790M) and HCC827-vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI-H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL-resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation- or MET amplification-positive tumors as long as their growth remained significantly suppressed by ERL., (© 2015 UICC.)

We demonstrate that an asymmetric composite cluster, [Ag25{C≡CC(CH3)3}16(CH3CN)4(P2W15Nb3O62)] (1), consisting of directly fused polyoxometalate and silver alkynide moieties can be facilely synthesized by a one-pot reaction between a Nb-substituted Dawson-type polyoxometalate, H4[α-P2W15Nb3O62](5-), and the mixture of (CH3)3CC≡CAg and CF3SO3Ag. Single-crystal X-ray diffraction revealed the structure of 1, where Ag atoms are selectively attached to the Nb-substituted hemisphere of the pedestal Dawson anion. Its structural integrity in the solution was demonstrated by (31)P NMR spectroscopy and analytical ultracentrifugation. The latter method also unveiled the stepwise formation mechanism of 1.