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Recent developments in conversational technologies have attracted researchers to study their applications in recommending items through conversations. It is considered that physical robots, rather than virtual ones, are effective in situations in which robots talk about items near participants. However, in real situations, robots may be required to recommend items that are present but invisible in the scene of the communication. In this study, we conducted a field experiment in a shopping mall to investigate the effects of robot embodiment on recommendation tasks. The robots recommended a dish after talking to participants about their food preferences. We developed a conversational recommendation system and implemented it using physical and virtual robots. The field experiment was conducted in a shopping mall; the visitors were encouraged to participate. The experiment lasted a total of 99 hours (9 hours per day for 11 days) inside and in front of a food court. Although no significant difference in the behavioral aspect was confirmed, the results obtained from 272 conversations suggested that having physical bodies enhanced the satisfaction and agreement with the robots' recommendations.

Takashi Miyachi,1 Kyoko Nomura,2 Sachiko Minamizono,2 Kazuki Sakai,1 Toyoto Iwata,2 Yuta Sugano,1 Shun Sawaguchi,3 Kouhei Takahashi,3 Kazuo Mishima4 1Department of Medicine, Akita University Graduate School of Medicine, Akita, Japan; 2Department of Environmental Health Science and Public Health, Akita University Graduate School of Medicine, Akita, Japan; 3Japan Health Insurance Association, Akita Branch, Akita, Japan; 4Department of Neuropsychiatry, Akita University Graduate School of Medicine, Akita, JapanCorrespondence: Kyoko NomuraDepartment of Environmental Health Science and Public Health, Akita University Graduate School of Medicine, 1– 1– 1 Hondo, Akita, 010-8543, JapanTel +81-18-884-6086Fax +81-18-836-2609Email nomurakyoko@gmail.comPurpose: Truck drivers with insomnia are at a high risk of traffic accidents. We aimed to investigate the prevalence of insomnia and identify its associated factors among truck drivers in Japan.Methods: Participants were 2927 male truck drivers younger than 65 years old. Self-administered questionnaires were used to assess insomnia symptoms, State-Trait Anxiety Inventory, drinking, smoking habits, body mass index, caffeine intake, as well as daily driving hours, consecutive days away from home, and driving distance. Insomnia symptoms included difficulty initiating sleep, maintaining sleep and early morning awakening. Insomnia was defined when any of these symptoms were observed with daily tiredness.Results: The prevalence of insomnia among the subjects was 13.3% (n=356), of which 13.5% had difficulty initiating sleep, 78% had difficulty maintaining sleep, and 26.4% had early morning awakening. After adjusting for covariates, drinking habits, daily driving hours, and STAI score were significantly and linearly associated with insomnia; the adjusted odds ratio (OR) of drinking habits for insomnia was 1.74 [95% confidence interval (CI), 1.23– 2.47] for heavy drinkers compared to non-drinkers (trend p< 0.001); the adjusted OR of daily driving hours was 1.87 (95% CI, 1.00– 3.49) for 12 hours or longer in a day compared to < 8 hours in a day (trend p< 0.001); the adjusted OR of STAI quartiles was 5.30 (95% CI, 3.66– 7.67) for the highest quartile compared to the lowest quartile (trend p< 0.001).Conclusion: The present study demonstrated that insomnia is prevalent among truck drivers in Japan, and its risk factors include drinking habits, daily driving hours, and anxiety.Keywords: insomnia, truck driver, State-Trait Anxiety Inventory, STAI, working environment, lifestyle

The rapid expansion of online job interviews during the COVID-19 pandemic is expected to continue after the pandemic has subsided. These interviews are a significant barrier for individuals with autism spectrum disorders (ASD). There is little evidence-based training for online job interviews for individuals with ASD, and the development of new trainings is expected. In an effort to facilitate online job interview skill acquisition for individuals with ASD, we developed a group-based online job interview training program using a virtual robot (GOT). In GOT, the interviewer and interviewee are projected as virtual robots on the screen. Five participants were grouped and performed the role of interviewee, interviewer, and evaluator. The participants performed all roles in a random order. Each session consisted of a first job interview session, feedback session, and second job interview session. The participants experienced 25 sessions. Before and after GOT, the participants underwent a mock online job interview with a human professional interviewer (MOH) to evaluate the effect of GOT. In total, 15 individuals with ASD took part in the study. The GOT improved self-confidence, motivation, the understanding of others' perspectives, verbal competence, non-verbal competence, and interview performance scores. There was also a significant increase in the recognition of the importance of the point of view of interviewers and evaluators after the second MOH compared to after the first MOH. Using a VR robot and learning the importance of interview skills by experiencing other perspectives (i.e., viewpoint of interviewer and evaluator) may have sustained their motivation and enabled greater self-confidence. Given the promising results of this study and to draw definitive conclusions regarding the efficacy of virtual reality (VR) robots for mock online job interview training, further studies with larger, more diverse samples of individuals with ASD using a longitudinal design are warranted.

In recent years, communication robots aiming to offer mental support to the elderly have attracted increasing attention. Dialogue systems consisting of two robots could provide the elderly with opportunities to hold longer conversations in care homes. In this study, we conducted an experiment to compare two types of scenario-based dialogue systems with different types of bodies—physical and virtual robots—to investigate the effects of embodying such dialogue systems. Forty elderly people aged from 65 to 84 interacted with either an embodied desktop-sized humanoid robot or computer graphic agent displayed on a monitor. The elderly participants were divided into groups depending on the success of the interactions. The results revealed that (i) in the group where the robots responded more successfully with the expected conversation flow, the elderly are more engaged in the conversation with the physical robots than the virtual robots, and (ii) the elderly in the group in which robots responded successfully are more engaged in the conversation with the physical robots than those in the group in which the robots responded with ambiguous responses owing to unexpected utterances from the elderly. These results suggest that having a physical body is advantageous in promoting high engagement, and the potential advantage appears depending on whether the system can handle the conversation flow. These findings provide new insight into the development of dialogue systems assisting elderly in maintaining a better mental health.

Through numerical simulations, it is predicted that the gravitational waves (GWs) reflect the characteristics of the core-collapse supernova (CCSN) explosion mechanism. There are multiple GW excitation processes that occur inside a star before its explosion, and it is suggested that the GWs originating from the CCSN have a mode for each excitation process in terms of time-frequency representation. Therefore, we propose an application of the Hilbert-Huang Transform (HHT), which is a high-resolution time-frequency analysis method, to analyze these GW modes for theoretically probing and increasing our understanding of the explosion mechanism. The HHT defines frequency as a function of time, and is not bound by the trade-off between time and frequency resolutions. In this study, we analyze a gravitational waveform obtained from a three-dimensional general-relativistic CCSN model that showed a vigorous activity of the standing-accretion-shock-instability (SASI). We succeed in extracting the SASI induced GWs with high resolution on a time-frequency representation using the HHT and we examine their instantaneous frequencies., Comment: 12 pages, 9 figures

Ringdown gravitational waves of compact binary mergers are an important target to test general relativity. The main components of the ringdown waveform after merger are black hole quasinormal modes. In general relativity, all multipolar quasinormal modes of a black hole should give the same values of black hole parameters. Although the observed binary black hole events so far are not significant enough to perform the test with ringdown gravitational waves, it is expected that the test will be achieved in third generation detectors. The Japanese gravitational wave detector KAGRA, called bKAGRA for the current configuration, has started observation, and discussions for the future upgrade plans have also started. In this study, we consider which KAGRA upgrade plan is the best to detect the subdominant quasinormal modes of black holes in the aim of testing general relativity. We use a numerical relativity waveform as injected signals that contains two multipolar modes and analyze each mode by matched filtering. Our results suggest that the plan FDSQZ, which improves the sensitivity of KAGRA for broad frequency range, is the most suitable configuration for black hole spectroscopy.

Mines are believed to have suitable cost performance because cheap weapons prevent opponents from freely engaging in activity in mined areas for a comparatively long time. Mine warfare (MW) can be regarded as combat between a minefield planner and mine countermeasure (MCM) forces. The minefield planner equips influence mines with mine counter-countermeasures (MCCMs), which increase the difficulty of their removal. One of the MCCMs is a mine shipcounter, which provides a mine detonation fuse that triggers with some delay after the mine sensor detects vessels. In this paper, we develop a mathematical model of the MCM operation against scattered naval mines with shipcounters and analyze MW as a two-sided warfare game between the minefield planner and the MCM forces. The MCM forces must make a clever choice between mine-hunting or sweeping operations because, depending on the MCM operations, the shipcount has different effects on the removal of mines or on the damage risk to civilian ships transiting in a channel. First, we evaluate the performances of hunting and sweeping considering the mine shipcount; second, we consider a simple game with the minefield planner and the MCM forces as players and analyze rational strategies for these decision makers in MW.

In order to understand the flow structure of a flickering flame, time-resolved observations are carried out of the velocity and density fields in a diffusion flame in co-flow using particle image velocimetry (PIV) and shadowgraph imaging, respectively. The PIV measurement combined with invalid velocity vector analysis using proper orthogonal decomposition (POD) allows for the high spatial resolution measurement of the velocity field in the flame, while the shadowgraph images show the high-temperature contour and soot formation of the flame. These experimental results indicate a periodic variation in the flow field and soot structure of the flickering flame. The observation of the velocity field shows that the periodic inflow and outflow are generated in the outer layer of the flame near the burner, which promotes the flow acceleration in the vertical direction leading to clip-off behavior. It is found that the vorticity contour evaluated from the velocity field matches well with the high-temperature contour observed in the shadowgraph images. Further examination of the flow structure is carried out using the first two POD modes, which demonstrate the presence of counter-rotating vortices in the outer flame contributing to stretching of the flame due to the clip-off motion in the flame.

In the development of lithium-air batteries, to understand how the porous structure of the cathode affects the performance of the battery system is important because the main electrochemical reaction which occurs in this system is the deposition of Li2O2 in the pores of the cathode. To clarify the relationship between the porous structure of the cathode and the performance of the battery system, we used carbon gels with different pore size distributions as model cathodes and investigated how their pores are filled during discharging by analyzing the structure of cathodes discharged to different depths through N-2 adsorption experiments. The dominant deposit formed during discharging was identified to be Li2O2 by X-ray diffraction. Interestingly, the ratio of the volume of the Li2O2 deposited in fully discharged cathodes to the meso-macropore volume of the cathode was in the range of 0.5-0.6 and was fairly constant among the samples. Such ratios are higher than those of microporous carbon cathodes such as activated carbon cathodes. These results suggest that increasing the meso-macro pore volume of the cathode is effective to obtain large capacities in lithium-air batteries.

We have been constructing a swimming ability improvement support system. One of the issues to be addressed is the automatic classification of swimming styles (backstroke, breaststroke, butterfly, and front crawl). The mainstream swimming style classification technique of conventional researches is based on non-ensemble learning; in their classification, breaststroke and butterfly are mixed up with each other. To improve its generalization performance, we need to use better classifiers and more adaptive feature values than previously considered. Therefore, this research has introduced (1) random forest technique, one of ensemble learning techniques, and (2) feature values specific to breaststroke and butterfly to construct a four-swimming-style classifier that has resolved this issue. From subjects with 7 to 20 years history of swimming races, we have obtained their sensor data during swimming and have divided the data into learning data and test data. We have also converted them into feature values that represent their body motions. We have selected from those body-motion-representing feature values the important data to classify four swimming styles and feature values specific to breaststroke and butterfly. We have used the learning data to construct a swimming style classifier, and the test data to evaluate its classification accuracy. The evaluation results show that (1’) the introduction of ensemble learning has improved the mean value of F-measure for breaststroke and butterfly by 0.053, and (2’) the introduction of feature values specific to breaststroke and butterfly has improved the mean value of F-measure for breaststroke and butterfly by 0.121 as compared with (1’). The proposed swimming style classifier has performed a mean F-measure of 0.981 for the four swimming styles as well as good classification accuracies for front crawl and backstroke. Therefore, we have concluded that the swimming style classifier we have constructed has resolved the problem of mixing up breaststroke and butterfly, as well as can properly classify all different swimming styles.

This paper describes a method of rule extraction for generation of appropriate actions by a robot in a multiparty conversation based on the relative probability of human actions in a similar situation. The proposed method was applied to a dataset collected from multiparty interactions between two robots and one human subject who took on the role of supporting one robot. By computing the relative occurrence probabilities of human actions after the execution of the robots’ actions, twenty rules describing human behavior in such a role were identified. To evaluate the rules, the human role was filled by a new bystander robot and other subjects were asked to report their impressions of video clips in which the bystander robot acted or did not act in accordance with the rules. The reported impressions and a quantitative analysis of the rules suggest that the behavior of listening and the supporting role that the subjects play can be reproduced by a bystander robot acting in accordance with the rules identified by the proposed method.

The ringdown part of gravitational waves in the final stage of merger of compact objects tells us the nature of strong gravity which can be used for testing the theories of gravity. The ringdown waveform, however, fades out in a very short time with a few cycles, and hence it is challenging for gravitational wave data analysis to extract the ringdown frequency and its damping time scale. We here propose to build up a suite of mock data of gravitational waves to compare the performance of various approaches developed to detect quasi-normal modes from a black hole. In this paper we present our initial results of comparisons of the following five methods; (1) plain matched filtering with ringdown part (MF-R) method, (2) matched filtering with both merger and ringdown parts (MF-MR) method, (3) Hilbert-Huang transformation (HHT) method, (4) autoregressive modeling (AR) method, and (5) neural network (NN) method. After comparing their performance, we discuss our future projects., Comment: 15 pages, 4 figures

To provide a better discussion experience in current argumentative dialogue systems, it is necessary for the user to feel motivated to participate, even if the system already responds appropriately. In this paper, we propose a method that can smoothly introduce argumentative dialogue by inserting an initial discourse, consisting of question-answer pairs concerning personality. The system can induce interest of the users prior to agreement or disagreement during the main discourse. By disclosing their interests, the users will feel familiarity and motivation to further engage in the argumentative dialogue and understand the system’s intent. To verify the effectiveness of a question-answer dialogue inserted before the argument, a subjective experiment was conducted using a text chat interface. The results suggest that inserting the question-answer dialogue enhances familiarity and naturalness. Notably, the results suggest that women more than men regard the dialogue as more natural and the argument as deepened, following an exchange concerning personality.

To improve the performance of carbon paper used for applications such as electrodes for electrochemical devices and air filters, two types of long carbon nanofibers (CNFs) with average diameters of 20 and 49 nm were prepared by the liquid pulse injection (LPI) technique by adjusting reaction conditions. Carbon paper was made from the CNFs through a simple filtration process. The paper prepared from the CNFs with an average diameter of 20 nm (LPI-CNF(20) paper) was firm and flexible even though it was prepared without using any binders. LPI-CNF(20) paper also had a high surface area and showed a high electrical conductivity and a moderate gas permeability according to its void size. These properties are required for cathodes in the latest battery systems such as lithium–air batteries. In electrochemical experiments conducted to evaluate the performance of LPI-CNF(20) paper as a cathode, the paper showed a larger discharge capacity on the basis of the cathode weight than a conventional cathode (a commercially available carbon paper combined with a porous carbon), which indicated that it has a high potential to be used as a cathode in lithium–air batteries.

It is known that a quasinormal mode (QNM) of a remnant black hole dominates a ringdown gravitational wave (GW) in a binary black hole (BBH) merger. To study properties of the QNMs, it is important to determine the time when the QNMs appear in a GW signal as well as to calculate its frequency and amplitude. In this paper, we propose a new method of estimating the starting time of the QNM and calculating the QNM frequency and amplitude of BBH GWs. We apply it to simulated merger waveforms by numerical relativity and the observed data of GW150914. The results show that the obtained QNM frequencies and time evolutions of amplitudes are consistent with the theoretical values within 1% accuracy for pure waveforms free from detector noise. In addition, it is revealed that there is a correlation between the starting time of the QNM and the spin of the remnant black hole. In the analysis of GW150914, we show that the parameters of the remnant black hole estimated through our method are consistent with those given by LIGO and a reasonable starting time of the QNM is determined., 12 pages, 12 figures

Galectins comprise a family of animal lectins that differ in their affinity for β-galactosides. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently shown to function as a ligand for T-cell immunoglobin domain and mucin domain-3 (Tim-3) expressed on terminally differentiated CD4(+) Th1 cells. Gal-9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal-9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal-9 inhibit MLR in a dose-dependent manner, involving both Ca(2+) influx and apoptosis in T cells. Next, we revealed that recombinant human Gal-9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal-9 ameliorates aGVHD, possibly by inducing T-cell apoptosis, suggesting that gal-9 may be an attractive candidate for the treatment of aGVHD.

An internal soliton in two-layered stratified fluids has been investigated by means of flow visualization. The relationship between the phase speed of soliton and its amplitude has been obtained experimentally. It has been also found that steady waves form behind the internal soliton traveling alongthe interface of a stratified fluid. The waves are of similar characteristics tothe lee waves which are observed behind a mountain in stratified atmosphericflow.

Although novel agents effective against malignant mesothelioma (MM) have been developed, the prognosis of patients with MM is still poor. We generated a DNA-chimeric siRNA against polo-like kinase-1 (PLK-1), which was more stable in human serum than the non-chimeric siRNA. The chimeric PLK-1 siRNA inhibited MM cell proliferation through the induction of apoptosis. Next, we investigated the effects of zoledronic acid (ZOL) on MM cells, and found that ZOL also induced apoptosis in MM cells. Furthermore, ZOL augmented the inhibitory effects of the PLK-1 siRNA. In conclusion, combining a PLK-1 siRNA with ZOL treatment is an attractive strategy against MM.

Effects of magnetization on the complex modulus of kappa-carrageenan magnetic gels have been investigated. The magnetic gel was made of a natural polymer, kappa-carrageenan, and a ferrimagnetic particle, barium ferrite. The complex modulus was measured before and after magnetization of the gel by dynamic viscoelastic measurements with a compressional strain. The gels showed a giant reduction in the storage modulus of approximately 10(7) Pa and also in the loss modulus of approximately 10(6) Pa due to magnetization. The reduction increased with increasing volume fraction of ferrite, and it was nearly independent of the frequency. It was also found that the change in the modulus was nearly independent of the magnetization direction and irradiation time of the magnetic fields to the gel. The magnetic gels demonstrating the giant reduction in the dynamic modulus showed a large nonlinear viscoelastic response. It was observed that the magnetic gel was deformed slightly due to magnetization. The observed giant complex modulus reduction could be attributed to the nonlinear viscoelasticity and deformation caused by magnetization. Magnetism, nonlinear viscoelasticity, and effects of magnetization on the morphological and shape changes were discussed.

Inactivation of bacteriophage by pulsed electric field (PEF) treatment, one of the effective procedures for bacteria nonthermal inactivation, was studied. Model phage particles Escherichia coli bacteriophages M13mp18 and λ phage, were successfully inactivated by PEF treatment. The survival ratios of both bacteriophages decreased depending on the PEF treatment time when applied peak voltage was 5 or 7 kV, and the survival ratios after 12 min PEF treatment were 10−4 – 10−5. Electrophoresis analyses of biological molecules of inactivated λ phage detected no degradation of total protein and genomic DNA. These results suggested that the factor of phage inactivation by PEF treatment was not based on the degradation of protein or DNA, but on the destruction of phage particle structure. Sensitivity of E. coli phage to PEF treatment was compared with that of E. coli cell. Phage and MV1184 cell were treated with same condition PEF at 5 kV, respectively. After 12 min treatment, the survival ration of λ phage and MV1184 were 4.0 × 10−5 and 1.7 × 10−3, respectively. The survival ratio of phage was lower than that of MV1184. E. coli cell is more tolerant to inactivation with PEF treatment than coli phage.

Abstract 2590 Poster Board II-566 Chronic myelogenous leukemia (CML) is caused by a consistent genetic abnormality, termed the Philadelphia chromosome (Ph). It results in the production of BCR-ABL fusion protein, a constitutively active tyrosine kinase. Imatinib mesylate (IM, Gleevec®), the first generation tyrosine kinase inhibitor (TKI), has revolutionized therapy for CML patients. However, resistance for IM develops in a significant proportion of cases, and is predominantly mediated by single point mutations within the BCR-ABL kinase domain. Second generation TKIs such as dasatinib (Sprycel®) and nilotinib (Tasigna®) represent viable alternatives for IM-resistant or intolerant CML patients. Each mutated BCR-ABL has different sensitivity to those TKIs. Thus, it is significantly important to detect early the existence of BCR-ABL mutations and their specificities in treating Ph+ leukemias. We have developed a novel automated method that has high sensitivity to detect a few copies of mutation sequences that are mixed in many copies of normal sequences. This method consists of PCR amplification step and Tm (melting temperature) analysis step that uses a quenching probe. And we have already shown that this system has clinical efficacy in JAK2V617F mutation that is one of the genetic hallmarks of chronic myeloproliferative diseases. (Tanaka R, et al. Leuk Res, 2008). When a whole blood sample or a purified DNA sample reacts with reagents, PCR and Tm analysis automatically processed in the same tube, and whole procedure finishes in approximately 1 hour. The detection of mutation is extremely accurate because the quenching probe is designed perfectly matched for mutated sequence. As Tm value of mutation sequence is higher than that of normal one, it is easy to detect the existence of mutation from the Tm analysis data. We have constructed the probes for 14 mutations concerned for IM-resistance (M244V, G250E, Q252H, Y253F, Y253H, E255V, E255K, T315I, T315A, F317L, M351T, E355G, F359V, and H396R). Considering the clinical significance of T315I mutation, which renders resistance to all currently available TKIs, we refined this method to higher sensitivity for detecting T315I mutation. First, we analyzed the sensitivity of this system on BCR-ABL. In dilution assays using wt and mutated plasmid, the system reliably quantified the mutation in a population containing as few as 3.0% mutant. Moreover, for T315I setting, we successfully detected as few as 0.3% (30 copies from 10,000 copies) mutations by a higher-sensitive assay. Next, we examined the clinical samples. Each sample was also examined by direct sequencing in comparison to our method. Kinase domain mutations were identified in 24 of the 50 (48%) patients. Our automated analysis was enabled to detect mutations in 19 patients, including p-loop mutations (G250E: n=3; E255K: n= 5), IM-binding domain mutations (T315I: n=10), and an activation-loop mutation (H396R: n=1). And all the positive cases (19 of 19) showed a concordance with the result of direct sequencing. On the other hand, 5 cases were detected just by direct sequencing, but all that cases were out of our setting mutations (Q252E, V379I, S417F, E459K). Impressively, in one case, only higher-sensitivity assay could reveal T315I mutation, although it was detected as a wild type both by direct sequence and our usual method. It suggests that the higher-sensitive system could detect low amount of T315I mutation in the earlier stage of disease. In conclusion, sensitivity of our system (3%) is significantly greater than that of direct sequencing (15 – 25%), and results can be obtained within one hour. By the serial monitoring, it is demonstrated the availability of the higher-sensitive analysis (0.3%) to detect T315I mutation. This rapid and accurate detection of clinically significant mutations enables us to contribute to better clinical practice in treating Ph+ leukemia patients, such as in selecting alternative strategies of IM dose escalation, second generation TKIs, or allogeneic stem cell transplantation. Disclosures: No relevant conflicts of interest to declare.

Abstract 1338 Poster Board I-360 Galectins are a family of soluble animal lectins that differ in their affinity for b-galactosides. Fifteen members of the human galectin family have been described to date. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently found to serve as a ligand for T cell immunoglobulin and mucin domain-3 (Tim-3), which is a Th1-specific type 1 membrane protein. Gal-9 modulates immune reactions, such as induction of apoptosis in Th1 cells. We herein investigated the effects of Gal-9 treatment on acute graft-versus-host disease (aGVHD) in a murine model. First, we assessed whether recombinant human (rh) Gal-9 can inhibit the mixed lymphocyte reaction (MLR) by culturing irradiated (30 Gy) splenic cells from 7- to 8-week-old female BDF1 mice with splenic cells from 7- to 8-week-old female C57BL/6J mice in the presence of various concentrations of Gal-9 for 10 days. rhGal-9 inhibited MLR in a dose-dependent manner. We then studied whether this effect was mediated by rhGal-9-induced apoptosis by culturing splenic cells from BDF1 mice with plate-bound anti-CD3 monoclonal antibody and Gal-9. Flow cytometric analysis revealed that rhGal-9 increased the number of Annexin-V+ cells in a dose-dependent manner (Figure. 1A). Thus, rhGal-9 inhibited MLR by inducing splenic cell apoptosis. This suppressive effect of Gal-9 on MLR was inhibited by lactose but not by sucrose (Figure. 1B). Taken together, Gal-9 induces T cell apoptosis through Ca2+ influx induced by binding to b-galactoside, resulting in the suppression of MLR. We then assessed whether rhGal-9 treatment altered aGVHD. Recipient B6D2F1 mice received a myeloablative dose (9 Gy) of total body irradiation from an X-ray irradiator. Six to eight hours later, each recipient mouse was injected i.v. with 4 × 106 TCD-BM cells and 5 × 106 mononuclear splenocytes. aGVHD clinical scores were evaluated once or twice a week. After aGVHD developed, recipient mice were treated with rhGal-9 (30 mg/mouse) or vehicle by i.p. injection for 14 consecutive days. The administration of rhGal-9 significantly attenuated aGVHD as compared to vehicle-treated mice (Figure. 2). Histological analyses also confirmed that aGVHD was declined by rhGal-9 treatment. Additionally, we investigated the effects of Gal-9 treatment on different T ell subsets. To analyze the effect of Gal-9 on donor lymphocytes, splenic mononuclear cells from GFP Tg mice were used for the induction of aGVHD. The gating parameter was first set to isolate the lymphocyte population of peripheral blood leukocytes, and then set for GFP+ cells. Gal-9 treatment decreased the frequency of CD4+/Tim-3+ cells and CD8+/intracellular IFN-g+ cells, whereas CD4+/CD25+ and CD25+/Foxp3+ Treg cells were increased by rhGal-9 treatment. These results suggest that Gal-9 regulates aGVHD by increasing regulatory T cells. In conclusion, Gal-9 ameliorates aGVHD by inducing T-cell apoptosis and also by increasing regulatory T cells. Disclosures No relevant conflicts of interest to declare.

Abstract 4245 We have previously reported that zoledronic acid (ZOL) had the anti-tumor activity against chronic myelogenous leukemia in the murine models and it could induce apoptosis to leukemic cells (Kuroda, et al. Blood, 2003). ZOL is a widely used agent to treat osteoporosis or hypercalcemia of malignancy because of its activity to induce apoptosis to osteoclasts (OCs), which are specialized cells to resorb the bone matrix. In this study, we hypothesized that OCs might be involved in the survival or the proliferation of leukemic cells in the bone marrow (BM) microenvironment which consists of osteoblasts (OBs), OCs, blood vessels, and so on. In order to verify our hypothesis, we used the in vitro co-culture system. Ba/F3 wt bcr-abl cells were indirectly co-cultured with the murine OBs, OCs, and/or the bone slices derived from the murine femoral bones in the transwell chambers. OBs and OCs enhanced the proliferation of Ba/F3 wt bcr-abl cells. Interestingly, OCs significantly suppressed the proliferation of Ba/F3 wt bcr-abl cells in the presence of the bone slices. Next, we examined the cell cycle status of Ba/F3 wt bcr-abl cells co-cultured in each condition. Ki-67-negative populations were reported to be in G0 phase, thus 7-AAD and Ki-67 double staining can distinguish population in G0 phase from that in G1 phase. It was revealed that OCs with bone slices increased the Ki-67-negative dormant populations in co-cultured Ba/F3 wt bcr-abl cells. Recently, it has been reported that TGF-β1 is a candidate cytokine that maintain the hibernation state of hematopoietic stem cells in the BM niche. Furthermore, TGF-β1 is one of the major cytokines accumulated in the mineralized bone matrix. Thus we evaluate the TGF-β1 levels in the culture supernatants of OBs, OCs, and/or bone slices by ELISA. The concentrations of TGF-β1 were significantly higher in the supernatants of OBs, OCs, and bone slices, thus it might be suggested that TGF-β1 was released from the bone slices by bone-resorbing OCs (Figure). Furthermore, TGF-β-neutralizing antibody reduced Ki-67-negative dormant populations in Ba/F3 wt bcr-abl cells co-cultured with OBs, OCs, and bone slices. In conclusion, our in vitro data demonstrate that OCs might maintain the dormant population of leukemic cells in the BM microenvironment and this effect was yielded, in part, by TGF-β1 released from the bone matrix by OCs. It was also suggested that OCs could be an effective target of a novel therapy against minimal residual disease of leukemia, which are thought to be resulted from the dormant population of leukemic cells residing in the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

We have previously reported that zoledoronic acid (ZOL) augmented the in vivo effect of imatinib in a murine chronic myeloid leukemia (CML) model (Blood 2003). ZOL alone induces apoptosis in leukemic cells in vitro by inhibiting prenylation of the Ras-related proteins. In addition to this direct anti-leukemic effect, we hypothesized that ZOL also has some influence in leukemic cells in vivo indirectly by destroying osteoclasts (OCs), which is the primary therapeutic activity of ZOL in osteoporosis patients. Supporting this notion is that by mediating bone resorption, OCs release a variety of cytokines such as IGF- 1, TGF-β, etc. that have accumulated in the bone matrix. It has been reported that OCs play an important role in bone metastasis of solid tumor, especially in cancer stem cells. However, little is known about the role of OCs in leukemia. Therefore, we investigated it in vitro and in vivo. For this purpose, we established an in vitro osteoblasts (OBs) and OCs co-culture system. The stable co-culture system that we developed includes collagen gel and murine primary OBs and OCs. In addition, murine femoral bone sections were sometimes added to this culture system so that the OCs could release the cytokines from the bone matrix. Thus, the collagen gel and OBs were placed in 12-well plates with and without bone sections and/or OCs. The transwell chambers over the wells then received 1×104 Ba/F3 cells that had been transfected with wild type bcr-abl (Ba/F3/bcr-abl cells). OBs markedly enhanced the growth of Ba/F3/bcr-abl cells in this indirect contact coculture system whereas the presence of both OBs and OCs slightly suppressed cell growth. Intriguingly, when bone sections were added (OBs+OCs+bone), Ba/F3/bcr-abl cell proliferation was significantly suppressed compared to the effect of OBs alone or OBs+OCs (Figure). Cell cycle analysis revealed that the G0/G1 population was increased in Ba/F3/bcr-abl cells co-cultured with OBs+OCs+bones. We also observed that the p27 protein levels of Ba/F3/bcr-abl cells increased upon co-culture with OCs or OCs+bones, similar to their response to treatment with purified TGF-β. We performed ELISAs to determine the concentrations of cytokines in the supernatants of co-cultured OBs and OCs. There were higher levels of TGF-β1 in the OBs+OCs+bones supernatant than in the OBs+OCs supernatant. Furthermore, OBs produced high levels of IGF-1. These findings suggest that OBs and OCs affect the proliferation and the cell cycle arrest of leukemic cells by releasing soluble factors, respectively. To more comprehensively elucidate the roles OCs play in leukemia cells in vivo, we used reveromycin A (RM-A) which inhibits bone resorption by specifically inducing apoptosis in OCs (Woo et al, PNAS 2006). RM-A did not have any in vitro effects on the proliferation of Ba/F3/bcr-abl cells. Thus, we could know the unalloyed role of OCs in leukemia with RM-A compared with ZOL which inhibited directly both OCs and leukemic cells. Our preliminary data show that RM-A suppresses the engraftment of inoculated Ba/F3/bcr-abl cells to nude mice. We also present data from ongoing studies showing the effect of RM-A on leukemic cells in murine models. These findings suggested that OCs may be an important constituent of leukemia stem cell niche and destruction of OCs by either ZOL or RM-A is a novel strategy for leukemia treatment. Figure Figure

During an examination of components contributing to the bitter taste of asparagus bottom cut (Asparagus officinalis L.), two new furostanol saponins were isolated from roots extractives. Their chemical structures were established as 5β-furostane-3β,22,26 triol-3-O-β-d-glucopyranosyl (1→2)-β-d-glucopyranoside 26-O-β-d-glucopyranoside and 5β-furostane-3β,22,26 triol-3-O-β-d-glucopyranosyl (1→2) [β-d-xylopyranoxyl (1→4)]-β-d-glucopyranoside 26-O-β-d-glucopyranoside respectively.