Maitake Beta-Glucan Enhances the Therapeutic Effect of Trastuzumab via Antibody-Dependent Cellular Cytotoxicity and Complement-Dependent Cytotoxicity.

Trastuzumab, an anti-HER2 monoclonal antibody, is the mainstay treatment for of HER2-positive breast cancer. However, trastuzumab resistance is often observed during treatment. Therefore, new therapeutic strategies are needed to enhance the clinical benefits of trastuzumab. Maitake ß-glucan MD-Fraction, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. In this study, we examined the effect of MD-Fraction on trastuzumab treatment of HER2-positive breast cancer. MD-Fraction did not directly inhibit the survival of HER2-positive breast cancer cells, alone or in the presence of trastuzumab in vitro. In HER2-positive xenograft models, the combination of MD-Fraction and trastuzumab was more effective than trastuzumab alone. Peripheral blood lymphocytes and splenic natural killer cells isolated from BALB/c nu/nu mice treated with MD-Fraction showed enhanced trastuzumab-induced antibody-dependent cellular cytotoxicity (ADCC) ex vivo. MD-Fraction-treated macrophages and neutrophils did not show enhanced trastuzumab cytotoxicity in the presence of heat-inactivated serum, but they showed enhanced cytotoxicity in the presence of native serum. These results suggest that MD-Fraction-treated macrophages and neutrophils enhance trastuzumab-induced complement-dependent cellular cytotoxicity (CDCC). Treatment of HER2-positive breast cancer cells with MD-Fraction in the presence of trastuzumab and native serum increased C3a release and tumor cell lysis in a dose-dependent manner, indicating that MD-Fraction enhanced trastuzumab-induced complement-dependent cytotoxicity (CDC) by activating the complement system. This study demonstrates that the combination of trastuzumab and MD-Fraction exerts a greater antitumor effect than trastuzumab alone by enhancing ADCC, CDCC, and CDC in HER2-positive breast cancer.

A secretory protein neudesin regulates splenic red pulp macrophages in erythrophagocytosis and iron recycling.

Neudesin, originally identified as a neurotrophic factor, has primarily been studied for its neural functions despite its widespread expression. Using 8-week-old neudesin knockout mice, we elucidated the role of neudesin in the spleen. The absence of neudesin caused mild splenomegaly, shortened lifespan of circulating erythrocytes, and abnormal recovery from phenylhydrazine-induced acute anemia. Blood cross-transfusion and splenectomy experiments revealed that the shortened lifespan of erythrocytes was attributable to splenic impairment. Further analysis revealed increased erythrophagocytosis and decreased iron stores in the splenic red pulp, which was linked to the upregulation of Fcγ receptors and iron-recycling genes in neudesin-deficient macrophages. In vitro analysis confirmed that neudesin suppressed erythrophagocytosis and expression of Fcγ receptors through ERK1/2 activation in heme-stimulated macrophages. Finally, we observed that 24-week-old neudesin knockout mice exhibited severe symptoms of anemia. Collectively, our results suggest that neudesin regulates the function of red pulp macrophages and contributes to erythrocyte and iron homeostasis.

Maitake α-glucan promotes differentiation of monocytic myeloid-derived suppressor cells into M1 macrophages.

AIMS: Myeloid-derived suppressor cells (MDSCs) are major components of the tumor microenvironment and systemically accumulate in tumor-bearing hosts and patients with cancer, facilitating cancer progression. Maitake macromolecular α-glucan YM-2A, isolated from Grifola frondosa, inhibits tumor growth by enhancing immune responses. The present study investigated the effects of YM-2A on the immunosuppressive potential of MDSCs. MAIN METHODS: YM-2A was orally administered to CT26 tumor-bearing mice, and the number of immune cells in the spleen and tumor was measured. Splenic MDSCs isolated from the CT26 tumor-bearing mice were treated with YM-2A and co-cultured with T cells to measure their inhibitory effect on T cell proliferation. For adoptive transfer of monocytic MDSCs (M-MDSCs), YM-2A-treated M-MDSCs mixed with CT26 cells were implanted subcutaneously in the mice to measure the tumor growth rate. KEY FINDINGS: YM-2A selectively reduced the accumulation of M-MDSCs but not that of polymorphonuclear MDSCs (PMN-MDSCs) in CT26 tumor-bearing mice. In tumor tissues, YM-2A treatment induced the polarity of immunostimulatory M1-phenotype; furthermore, it increased the infiltration of dendritic, natural killer, and CD4+ and CD8+ T cells. YM-2A treatment of purified M-MDSCs from CT-26 tumor-bearing mice induced dectin-1-dependent differentiation into M1 macrophages. YM-2A-treated M-MDSCs lost their inhibitory activity against proliferation and activation of CD8+ T cells. Furthermore, adoptive transfer of M-MDSCs treated with YM-2A inhibited CT26 tumor growth. SIGNIFICANCE: YM-2A promotes the differentiation of M-MDSCs into immunostimulatory M1 macrophages, thereby enhancing the efficacy of cancer immunotherapy.

Decrease in the T-box1 gene expression in embryonic brain and adult hippocampus of down syndrome mouse models.

Down syndrome (DS, Trisomy 21) is the most common genetic cause of delayed fetal brain development and postnatal intellectual disability. Although delayed fetal brain development might be involved in intellectual disability, no evidence of an association between these abnormal phenotypes has been shown. To identify molecules differentially expressed in both the prenatal forebrain and adult hippocampus of Ts1Cje mice, a mouse model of DS, we employed a transcriptomic analysis. In the present study, we conducted transcriptomic profiling of the hippocampus of adult Ts1Cje mice and compared the results with the previously obtained transcriptomic profile of the prenatal forebrain at embryonic day 14.5. Results showed that the Tbx1 mRNA expression was decreased at both life stages. In addition, the decreased expression of Tbx1 mRNA was confirmed in other DS mouse models, Dp(16)1Yey/+ and Ts1Rhr mice, which carry longer and shorter trisomic regions, respectively. Taken together, these findings suggest that Tbx1 may link the delayed fetal brain development and intellectual disability in DS.

Perturbation of the immune cells and prenatal neurogenesis by the triplication of the Erg gene in mouse models of Down syndrome.

Some mouse models of Down syndrome (DS), including Ts1Cje mice, exhibit impaired prenatal neurogenesis with yet unknown molecular mechanism. To gain insights into the impaired neurogenesis, a transcriptomic and flow cytometry analysis of E14.5 Ts1Cje embryo brain was performed. Our analysis revealed that the neutrophil and monocyte ratios in the CD45-positive hematopoietic cells were relatively increased, in agreement with the altered expression of inflammation/immune-related genes, in Ts1Cje embryonic brain, whereas the relative number of brain macrophages was decreased in comparison to wild-type mice. Similar upregulation of inflammation-associated mRNAs was observed in other DS mouse models, with variable trisomic region lengths. We used genetic manipulation to assess the contribution of Erg, a trisomic gene in these DS models, known to regulation hemato-immune cells. The perturbed proportions of immune cells in Ts1Cje mouse brain were restored in Ts1Cje-Erg+/+/Mld2 mice, which are disomic for functional Erg but otherwise trisomic on a Ts1Cje background. Moreover, the embryonic neurogenesis defects observed in Ts1Cje cortex were reduced in Ts1Cje-Erg+/+/Mld2 embryos. Our findings suggest that Erg gene triplication contributes to the dysregulation of the homeostatic proportion of the populations of immune cells in the embryonic brain and decreased prenatal cortical neurogenesis in the prenatal brain with DS.

Copper accumulation in the brain causes the elevation of oxidative stress and less anxious behavior in Ts1Cje mice, a model of Down syndrome.

Elevated oxidative stress (OS) is widely accepted to be involved in the pathogenesis of Down syndrome (DS). However, the mechanisms underlying the elevation of OS in DS are poorly understood. Biometals, in particular copper and iron, play roles in OS. We therefore focused on biometals in the brain with DS. In this study, we analyzed the profile of elements, including biometals, in the brain of Ts1Cje mice, a widely used genetic model of DS. An inductively coupled plasma-mass spectrometry (ICP-MS)-based comparative metallomic/elementomic analysis of Ts1Cje mouse brain revealed a higher level of copper in the hippocampus and cerebral cortex, but not in the striatum, in comparison to wild-type littermates. The expression of the copper transporter CTR1, which is involved in the transport of copper into cells, was decreased in the ependymal cells of Ts1Cje mice, suggesting a decrease in the CTR1-mediated transport of copper into the ependymal cells, which excrete copper into the cerebrospinal fluid. To evaluate the pathological significance of the accumulation of copper in the brain of Ts1Cje mice, we examined the effects of a diet with a low copper content (LoCD) on the elevated lipid peroxidation, the accumulation of hyperphosphorylated tau, and some behavioral anomalies. Reducing the copper concentration in the brain by an LoCD restored the enhanced lipid peroxidation and phosphorylation of tau in the brain and reduced anxiety-like behavior, but not hyperactivity or impaired spatial leaning, in Ts1Cje mice. The findings highlight the reduction of accumulation of copper in the brain may be a novel therapeutic strategy for DS.

Suppression of riboflavin-sensitized singlet oxygen generation by l-ascorbic acid, 3-O-ethyl-l-ascorbic acid and Trolox.

Riboflavin (RF), a water-soluble vitamin B2, is an endogenous singlet oxygen photosensitizer in human skin and eye. Time profiles of the near-infrared phosphorescence of singlet oxygen generated by RF have been measured in the absence and presence of l-ascorbic acid (AA, vitamin C), 3-O-ethyl-l-ascorbic acid (3-EtAA) and Trolox (TX, a water-soluble analogue of vitamin E) in phosphate buffer (pH 6.8). These substances suppress the RF-photosensitized singlet oxygen generation. For example, the quantum yield of singlet oxygen generation is decreased to a third by adding 0.4 mmol dm-3 AA or TX (the concentration of dissolved oxygen in air-saturated water is 0.27 mmol dm-3). AA and TX are more efficient suppressors of RF-photosensitized singlet oxygen generation than 3-EtAA. The bimolecular rate constants for quenching of the excited singlet and triplet states of RF by AA, 3-EtAA and TX have been determined through measurements of fluorescence and transient absorption. These measurements suggest that the observed suppression is due to the quenching of the excited singlet and triplet states of RF by AA, 3-EtAA and TX. The bimolecular rate constants for quenching of singlet oxygen by AA, 3-EtAA and TX were determined to be 1.8 × 108, 0.27 × 108, and 4.4 × 108 mol-1 dm3 s-1, respectively.

Photophysical properties of diethylhexyl 2,6-naphthalate (Corapan TQ), a photostabilizer for sunscreens.

Diethylhexyl 2,6-naphthalate (DEHN, tradename Corapan TQ) is used as a photostabilizer for a widely-used UV-A absorber, butylmethoxydibenzoylmethane (BMDBM). The photophysical properties of DEHN have been studied through measurements of the transient absorption, fluorescence, phosphorescence and EPR spectra in ethanol. The energy level of the lowest excited singlet state of DEHN is higher than that of BMDBM (enol form). The singlet-singlet energy transfer from BMDBM to DEHN is energetically unfavourable. The energy level of the lowest excited triplet (T1) state of DEHN is lower than that of BMDBM. It is probable that the mechanism of photostabilization of BMDBM by DEHN is the triplet-triplet energy transfer from BMDBM to DEHN. The observed phosphorescence spectrum, T1 lifetime and zero-field splitting parameters indicate that the T1 state of DEHN can be regarded mainly as a locally excited 3ππ* state within naphthalene. The sum of the quantum yields of fluorescence of DEHN (0.59 ± 0.09) and singlet oxygen generation by DEHN (0.44 ± 0.04) is almost unity in ethanol. About 60% of the UV energy absorbed by DEHN is released as fluorescence, while about 40% is used to produce the excited triplet state. The photoexcited states of 2-naphthoic acid have been studied for comparison.

Copper-Catalyzed Regioselective Hydroalkylation of 1,3-Dienes with Alkyl Fluorides and Grignard Reagents.

Copper complexes generated in situ from CuCl2, alkyl Grignard reagents, and 1,3-dienes play important roles as catalytic active species for the 1,2-hydroalkylation of 1,3-dienes by alkyl fluorides through C-F bond cleavage. The alkyl group is introduced to an internal carbon atom of the 1,3-diene regioselectively, thus giving rise to the branched terminal alkene product.

Copper-catalyzed alkyl-alkyl cross-coupling reactions using hydrocarbon additives: efficiency of catalyst and roles of additives.

Cross-coupling of alkyl halides with alkyl Grignard reagents proceeds with extremely high TONs of up to 1230000 using a Cu/unsaturated hydrocarbon catalytic system. Alkyl fluorides, chlorides, bromides, and tosylates are all suitable electrophiles, and a TOF as high as 31200 h(-1) was attained using an alkyl iodide. Side reactions of this catalytic system, i.e., reduction, dehydrohalogenation (elimination), and the homocoupling of alkyl halides, occur in the absence of additives. It appears that the reaction involves the ß-hydrogen elimination of alkylcopper intermediates, giving rise to olefins and Cu-H species, and that this process triggers both side reactions and the degradation of the Cu catalyst. The formed Cu-H promotes the reduction of alkyl halides to give alkanes and Cu-X or the generation of Cu(0), probably by disproportionation, which can oxidatively add to alkyl halides to yield olefins and, in some cases, homocoupling products. Unsaturated hydrocarbon additives such as 1,3-butadiene and phenylpropyne play important roles in achieving highly efficient cross-coupling by suppressing ß-hydrogen elimination, which inhibits both the degradation of the Cu catalyst and undesirable side reactions.

Consistent and robust determination of border ownership based on asymmetric surrounding contrast.

Determination of the figure region in an image is a fundamental step toward surface construction, shape coding, and object representation. Localized, asymmetric surround modulation, reported neurophysiologically in early-to-intermediate-level visual areas, has been proposed as a mechanism for figure-ground segregation. We investigated, computationally, whether such surround modulation is capable of yielding consistent and robust determination of figure side for various stimuli. Our surround modulation model showed a surprisingly high consistency among pseudorandom block stimuli, with greater consistency for stimuli that yielded higher accuracy of, and shorter reaction times in, human perception. Our analyses revealed that the localized, asymmetric organization of surrounds is crucial in the detection of the contrast imbalance that leads to the determination of the direction of figure with respect to the border. The model also exhibited robustness for gray-scaled natural images, with a mean correct rate of 67%, which was similar to that of figure-side determination in human perception through a small window and of machine-vision algorithms based on local processing. These results suggest a crucial role of surround modulation in the local processing of figure-ground segregation.

Spatial attention in early vision for the perception of border ownership.

Spatial attention alters contrast gain in early visual areas, which might affect the determination of border ownership (BO) that indicates the direction of figure with respect to the border. We investigated the role of spatial attention applied to early vision in the determination of BO with a computational model that consists of V1, V2, and posterior parietal (PP) modules. Attention alters contrast gain in the V1 module so that it enhances local contrast. The V2 module determines BO based on the surrounding contrast extracted by the V1 module. The simulation results showed that the attention significantly modulates BO; BO is even flipped in figures with ambiguous BO while BO is stable for unambiguous figures such as a simple square. To evaluate the model quantitatively, we carried out psychophysical experiments to measure the effects of attention in the perception of BO and compared the results with those from corresponding simulations. The model showed good agreement with human perception including the determination of BO for ambiguous random-block stimuli. These results indicate that the activity of BO-selective neurons could be modulated significantly by spatial attention that alters local contrast gain in V1, which may account in part for automatic, bi-stable perception in ambiguous figures.

Developmental competence of cat oocytes from ovaries stored at various temperature for 24 h.

We conducted two experiments to investigate the effects of storage temperature and period for cat ovaries on the meiotic and developmental competence of oocytes collected from the ovaries. In Experiment 1, ovaries were stored in physiological saline for 24 h at 4 C, 23-25 C, or 38 C (cold, room, and incubator temperature groups, respectively), and then oocytes were collected from the ovaries in each group. Morphologically intact oocytes were then selected and cultured in maturation medium for 24 h. Significantly more oocytes reached metaphase II (MII) in the cold temperature group (53.4%) than in the room and incubator temperature groups (20.0 and 2.4%, respectively). In Experiment 2, ovaries were stored in physiological saline at room temperature for 0, 6, 12 or 18 h, and then they were stored at 4 C (cold storage) until reaching a total storage period of 24 h. After storage of the ovaries, morphologically intact oocytes were matured, fertilized with frozen-thawed spermatozoa, and cultured in vitro. The rates of morphologically intact oocytes obtained from the ovaries stored at room temperature for 0, 6, 12 or 18 h were 35.3, 30.0, 26.4 and 14.7%, respectively, and the rates of intact oocytes that reached MII were 63.2, 36.4, 26.5 and 11.9%, respectively. The results suggested that the numbers of morphologically intact oocytes and intact oocytes that reached MII after in vitro maturation decrease gradually as the period of storage at room temperature before cold storage increases. Only oocytes from ovaries stored for 6 h developed to the blastocyst stage after in vitro maturation and fertilization when ovaries were stored at room temperature before cold storage. These results indicate that 24 h storage of ovaries at high temperatures (>23 C) decreases the meiotic competence of oocytes. The quality and developmental competence of oocytes are influenced by the length of storage at room temperature before cold storage.

Effects of electric field strengths on fusion and in vitro development of domestic cat embryos derived by somatic cell nuclear transfer.

The present study was conducted to determine the effect of electric field strength on the rate of membrane fusion between the somatic cell and cytoplast and on subsequent in vitro development of reconstructed embryos. Additionally, the in vitro developmental competence of cat oocytes artificially activated after 44 h of maturation culture was examined. An efficient fusion rate (64.2%) was obtained by applying a single pulse of 1.5 kV/cm for 50 micros, and the fusion rate remained almost constant at the higher field intensity (59.8 and 54.9% at 1.7 and 2.0 kV/cm, respectively). Although the cleavage rate of fused embryos increased with an increase of the electric field strength, there were no differences among the groups with respect to the proportion of development to the morula and blastocyst stages. In the additional experiment, oocytes at the metaphase II stage after culture for 44 h were activated by the combination of calcium ionophore (CaI) with cycloheximide (CHX). Some (11.8%) of activated oocytes developed to the blastocyst stage. Results from this study indicated that electric field strength affects the rates of fusion and cleavage but has no significant effects on the development to the blastocyst stage of reconstructed embryos. Prolonged maturation culture of cat oocytes (up to 44 h) decreased their ability to develop to the blastocyst stage.

Effects of single and double exposure to brilliant cresyl blue on the selection of porcine oocytes for in vitro production of embryos.

The aim of this study was to evaluate the effectiveness and toxicity of single and double application of the brilliant cresyl blue (BCB) test on the selection of porcine oocytes as an indirect measure of oocyte growth for in vitro fertilization (IVF) and nuclear transfer. In the first experiment, oocytes were exposed to BCB before and after maturation culture and classified according to their cytoplasmic coloration: blue coloration and colorless. The classified oocytes were fertilized with spermatozoa and then cultured for 7 days. The percentages of maturation to metaphase II in blue oocytes at the start of maturation culture were higher than those of colorless oocytes (68.7-70.1% versus 0.8-3.6%, P < 0.05). However, double application of BCB test before and after maturation culture had a toxic effect on fertilization and embryonic development. No significant differences in the blastocyst formation were found between blue oocytes without double application of BCB test and control oocytes without any application of BCB test, whereas the total cell number per blastocyst from the blue oocytes was higher than that from the control oocytes (48.0 versus 34.2, P < 0.05). In the second experiment, oocytes were exposed to the BCB test before or after maturation culture, and then the matured oocytes were activated to evaluate the ability of parthenogenetic development. The proportion of blastocyst formation of blue oocytes classified after maturation culture was lower than that of blue oocytes classified before maturation culture (10.0% versus 27.0%, P < 0.05). Therefore, double application of the BCB test before and after maturation culture impaired fertilization and embryonic development, whereas a single application before maturation culture was efficient to select oocytes for IVF and nuclear transfer.

Effect of maturation culture period of oocytes on the sex ratio of in vitro fertilized bovine embryos.

It has been suggested that the maturational stage of oocytes at time of insemination influences the sex ratio of resulting embryos. However, there are very few reports concerning the relationship between the maturation culture period of oocytes and the sex ratio of resulting embryos. The objective of this study was to investigate the effects of in vitro maturation culture period for bovine oocytes on the sex ratio of in vitro produced blastocysts using a novel technique of loop-mediated isothermal amplification (LAMP). Cumulus-oocyte complexes were collected from the ovaries of slaughtered cows, and then matured in vitro for various periods (16, 22, 28, and 34 h). After maturation culture for each period, the oocytes were inseminated with frozen-thawed spermatozoa, and then cultured in vitro. Blastocysts were harvested on Day 7 after insemination, and the sex of the embryos was examined using the LAMP method. The rates of oocytes matured to the metaphase II stage were significantly lower (P < 0.05) in the 16-h maturation group than in the other groups. The proportion of blastocyst formation after insemination was significantly higher (P < 0.05) in the 22-h maturation group than in the other groups. The proportion of male blastocysts increased with the increase in maturation culture period. The proportion of male blastocysts derived from oocytes matured for 34 h was significantly higher (P < 0.05) than from oocytes matured for 16 and 22 h. These results indicate that the sex ratio of in vitro fertilized embryos is apparently influenced by the maturation culture period of the oocytes.