Anticancer immunotherapy using inactivated Sendai virus particles

Ultraviolet-inactivated, replication-defective Sendai virus particles (Hemagglutinating virus of Japan envelope, HVJ-E) injected into murine colon carcinoma (CT26) tumors growing in syngeneic Balb/c mice eradicated 60-80% of the tumors and obviously inhibited the growth of the remainder. Induced adaptive anti-tumor immune responses were dominant in the tumor eradication process because the effect was abrogated in severe combined immunodeficient (SCID) mice. Murine and human dendritic cells (DCs) underwent dose-dependent maturation by HVJ-E in vitro. Profiles of cytokines secreted by DCs after HVJ-E stimulation showed that the amount of IL-6 released was comparable to that elicited by live HVJ. Real-time RT-PCR and immunohistochemistry revealed that HVJ-E induced a remarkable infiltration of DCs, CD4+ and CD8+ T cells into tumors and CT26 specific cytotoxic T lymphocytes (CTL) were induced. On the other hand, conditioned medium from DCs stimulated by HVJ-E (H-DCCM) rescued CD4+CD25- effector T cell proliferation from Foxp3+CD4+CD25+ regulatory T cell (Treg) mediated suppression and IL-6 was presumably dominant for this phenomenon. We also confirmed such rescue in mice treated with HVJ-E in vivo. Moreover, anti-tumor effect of HVJ-E was significantly reduced by an in vivo blockade of IL-6 signaling. Depending on cancer cell types, it is also expected that HVJ-E eradicates tumor by its direct cytotoxity against cancer cells or activating NK cells. Because it can enhance anti-tumor immunity and simultaneously remove Treg mediated suppression, HVJ-E shows promise as a novel therapeutic for cancer immunotherapy.     

Effects of a hop water extract on the compound 48/80-stimulated vascular permeability in ICR mice and histamine release from OVA-sensitized BALB/c mice

The antiallergic properties of a hop water extract (HWE) were studied by evaluating the Evans blue leakage from ICR mice caused by compound 48/80 stimulation, and the histamine release from ovalbumin (OVA)-sensitized BALB/c mice. An oral administration of HWE significantly inhibited the vascular permeability and histamine release. HWE itself did not have any influence on the total and antigen-specific immunoglobulin E (IgE) production in OVA-sensitized mice. These results indicate that HWE exerted an antiallergic effect by inhibiting the release of chemical mediators from mast cells and basophiles.     

A sensitive multiplex assay for piRNA expression

PIWI-interacting RNAs (piRNAs) are a new class of small RNAs specifically expressed in male germ cells. It is known to bind to PIWI class of Argonaute proteins, Mili and Miwi. To help to decipher the mechanism of piRNA function, here, we report a real time PCR-based multiplex assay for piRNA expression. Firstly, we showed that the assay specifically detects piRNA expression in adult testis, consistent with the Northern blot result. The method we developed can simultaneously detect at least eight piRNAs using only 10 pg total RNA, which is equivalent to the RNA present in a single cell. This is five to six order magnitude more sensitive than corresponding Northern blot assays. Finally we used this assay to analyze eight piRNAs expression in mouse primordial germ cells (PGCs) in genital ridges from E12.5, at the time when piRNA-binding protein Mili starts to be detected in PGCs. This multiplex piRNA assay can be further expanded to assay a few hundred of piRNAs simultaneously from as little as total RNA from a single cell. This approach will help to understand the mechanism and function of piRNAs during germ cell development.     

Sendai virus F glycoprotein induces IL-6 production in dendritic cells in a fusion-independent manner

We previously reported that inactivated Sendai virus particle (hemagglutinating virus of Japan envelope; HVJ-E) has anti-tumor effects by eliciting IL-6 production in dendritic cells (DCs). In the present study, we investigated which components of HVJ-E elicit IL-6 production. HVJ-E containing F0 protein inactive for virus envelope-cell membrane fusion enhanced IL-6 production. Reconstituted liposomes containing F protein stimulated IL-6 production. The antibody against F protein inhibited IL-6 secretion by HVJ-E. When carbohydrate chains of the F glycoprotein were removed, HVJ-E lost the ability to stimulate IL-6 secretion. These results suggest that F glycoprotein is required for IL-6 production in DCs.     

Intratumoral injection of inactivated Sendai virus particles elicits strong antitumor activity by enhancing local CXCL10 expression and systemic NK cell activation

We have already demonstrated that inactivated, replication-defective Sendai virus particles (HVJ-E) have a powerful antitumor effect by both the generation of tumor-specific cytotoxic T cells and inhibition of regulatory T cell activity. Here, we report that HVJ-E also has an antitumor effect through non-T cell immunity. Microarray analysis revealed that direct injection of HVJ-E induced the expression of CXCL10 in established Renca tumors. CXCL10 was secreted by dendritic cells in the tumors after HVJ-E injection. Quantitative real-time RT-PCR and immunohistochemistry revealed that CXCR3+ cells (predominantly NK cells) infiltrated the HVJ-E-injected tumors. Moreover, HVJ-E injection caused systemic activation of NK cells and enhanced their cytotoxity against tumor cells. In an in vivo experiment, approximately 50% of tumors were eradicated by HVJ-E injection, and this activity of HVJ-E against Renca tumors was largely abolished by NK cell depletion using anti-asialo GM1 antibody. Since HVJ-E injection induced systemic antitumor immunity by enhancing or correcting the chemokine-chemokine receptor axis, it might be a potential new therapy for cancer.     

Effects of temperature on the development and circannual control of pupation in the carpenter moth, <Emphasis Type="Italic">Cossus insularis</Emphasis> (Lepidoptera: Cossidae), reared on an artificial diet

The effects of temperature on larval development and the timing of pupation in the carpenter moth, Cossus insularis (Staudinger) (Lepidoptera: Cossidae) were examined by artificial rearing under different temperatures and the same photoperiod (15L:9D). Although C. insularis pupated and emerged at 20, 25, and 30 °C, the pupation rate was lower at 20 °C than at 25 and 30 °C. These results suggest that the optimum temperature range for preadult development is 25–30 °C. The duration of larval development was about 260 days for the first pupation group at 25 and 30 °C, and at least 600 days at 20 °C. Therefore, the C. insularis generation time was 2 years or more, as the total effective temperature for development from hatching to the pupal stage was unlikely to be reached within 1 year in Tokushima Prefecture. The second group pupated at 25 °C, about 200 days after the first group. This periodicity of pupation was likely due to the free-running period of the circannual rhythm. Furthermore, although only the first group pupated at 30 °C, the peak was almost synchronous with the first group at 25 °C. These results indicate that the timing of the first pupation group in C. insularis is temperature compensated. Therefore we propose that the presence of an endogenous rhythm during the development of C. insularis is evidence for a circannual rhythm related to the timing of pupation.     

Calofolic acids A–F,chromanones from the bark of Calophyllum scriblitifolium with vasorelaxation activity

Vasorelaxation activity guided separation of the methanol extract of Calophyllum scriblitifolium bark led to the isolation of 6 chromanones (calofolic acids A–F, 1–6). Their structures were elucidated by 1D and 2D NMR spectroscopy, and their absolute configurations were investigated by a combination of CD spectroscopy and DFT calculation. All isolated chromanones showed dose-dependent vasorelaxation activity on isolated rat aorta.     

Bone marrow-derived osteoblast progenitor cells in circulating blood contribute to ectopic bone formation in mice

Recent studies have suggested the existence of osteoblastic cells in the circulation, but the origin and role of these cells in vivo are not clear. Here, we examined how these cells contribute to osteogenesis in a bone morphogenetic protein (BMP)-induced model of ectopic bone formation. Following lethal dose-irradiation and subsequent green fluorescent protein-transgenic bone marrow cell-transplantation (GFP-BMT) in mice, a BMP-2-containing collagen pellet was implanted into muscle. Three weeks later, a significant number of GFP-positive osteoblastic cells were present in the newly generated ectopic bone. Moreover, peripheral blood mononuclear cells (PBMNCs) from the BMP-2-implanted mouse were then shown to include osteoblast progenitor cells (OPCs) in culture. Passive transfer of the PBMNCs isolated from the BMP-2-implanted GFP-mouse to the BMP-2-implanted nude mouse led to GFP-positive osteoblast accumulation in the ectopic bone. These data provide new insight into the mechanism of ectopic bone formation involving bone marrow-derived OPCs in circulating blood.