Non-glycosylated G protein with CpG ODN provides robust protection against respiratory syncytial virus without inducing eosinophilia

IntroductionRespiratory syncytial virus (RSV) vaccines targeting the fusion glycoprotein (F protein) are highly effective clinically in preventing RSV challenges. The attachment glycoprotein (G protein) is a potentially effective vaccine antigen candidate, as it is important for cell adhesion during infection. However, vaccine-associated enhanced diseases in mice, such as eosinophilic lung inflammation following RSV challenge, are a concern with G protein vaccines. This study aimed to design an effective G protein vaccine with enhanced safety and efficacy by evaluating the efficacy and adverse reactions of vaccines composed of different recombinant G proteins and adjuvants in mice.MethodsMice were subcutaneously immunized with glycosylated G protein expressed in mammalian cells (mG), non-glycosylated G protein expressed in Escherichia coli (eG), or F protein with or without aluminum salts (alum), CpG oligodeoxynucleotide (CpG ODN), or AddaVax. After vaccination, the levels of G-specific antibody and T-cell responses were measured. The immunized mice were challenged with RSV and examined for the viral load in the lungs and nasal turbinates, lung-infiltrating cells, and lung pathology.ResultsmG with any adjuvant was ineffective at inducing G-specific antibodies and had difficulty achieving both protection against RSV challenge and eosinophilia suppression. In particular, mG+CpG ODN induced G-specific T helper 1 (Th1) cells but only a few G-specific antibodies and did not protect against RSV challenge. However, eG+CpG ODN induced high levels of G-specific antibodies and Th1 cells and protected against RSV challenge without inducing pulmonary inflammation. Moreover, the combination vaccine of eG+F+CpG ODN showed greater protection against upper respiratory tract RSV challenge than using each single antigen vaccine alone.DiscussionThese results indicate that the efficacy of recombinant G protein vaccines can be enhanced without inducing adverse reactions by using appropriate antigens and adjuvants, and their efficacy is further enhanced in the combination vaccine with F protein. These data provide valuable information for the clinical application of G protein vaccines

G protein-coupled sphingosine-1-phosphate receptors: potential molecular targets for angiogenic and anti-angiogenic therapies

Sphingosine-1-phosphate (S1P) is a plasma lipid mediator with pleiotropic activities; it is constitutively produced in red blood cells and vascular endothelial cells through phosphorylation of sphingosine by one of two S1P synthesizing enzymes, sphingosine kinase 1 and 2 (SphK 1, 2), and exported into plasma to bind to high density lipoprotein and albumin. Sphingosine-1-phosphate acts through five members of the G protein-coupled S1P receptors (S1PR1-S1PR5) to exert diverse actions, which include vascular maturation in embryonic stage and postnatal angiogenesis, maintenance of functional integrity of vascular endothelium, regulation of vascular tonus, and lymphocyte trafficking. Sphingosine-1-phosphate is unique in its ability to regulate cell migration either positively or negatively by acting through different receptor subtypes. S1PR1 and S1PR3 mediate chemotactic cell migration toward S1P via Gi/Rac pathway, whereas S1PR2 mediates S1P inhibition of chemotaxis via G12/13/Rho-dependent inhibition of Rac. Sphingosine-1-phosphate positively or negatively regulates tumor cell migration, invasion in Matrigel, and hematogenous metastasis in manners strictly dependent on S1P receptor subtypes expressed in tumor cells. S1PR1 (and S1PR3) also mediates activation of Gi/phosphatidylinositol 3-kinase (PI3K)/Akt and stimulation of cell proliferation/survival, whereas S1PR2 could mediate suppression of cell proliferation/survival through G12/13/Rho/Rho kinase/PTEN-dependent Akt inhibition. S1PR1 (and S1PR3) expressed in endothelial cells mediates angiogenic action of S1P by stimulating endothelial cell migration, proliferation and tube formation. In a mouse model of hindlimb ischemia after femoral artery resection, repeated local administration or sustained delivery of S1P, or transgenic overexpression of SphK1, accelerates post-ischemic angiogenesis, through the S1P actions on both endothelial cells and bone marrow-derived myeloid cells (BMDCs). In tumor cells, SphK1 is upregulated especially in advanced stages, through mechanisms involving both activating Ras mutation and hypoxia, which leads to increased S1P production and also decreased cellular content of pro-apoptotic sphingolipid ceramide, a metabolic precursor of S1P. Apoptotic tumor cells also produce S1P through SphK2 activation, thus implicated in tumor angiogenesis by acting on endothelial cells through S1PR1/S1PR3, as well as tumor-infiltrating macrophages and BMDCs. Inhibition of S1PR1 function by either an anti-S1P antibody or FTY720 inhibits tumor angiogenesis and tumor growth. Differently from S1PR1, S1PR2 expressed in host cells mediates inhibition of tumor angiogenesis and tumor growth, through mechanisms involving the suppression of endothelial cell migration, proliferation and tube formation, and inhibition of BMDC recruitment to tumor stroma with suppressed expression of pro-angiogenic factor and matrix metalloprotease 9. These findings provide the molecular basis for S1P receptor subtype-selective targeting strategies aiming at angiogenic therapy for occlusive peripheral arterial diseases, and anti-angiogenic and anti-tumor therapies against cancer.Biomedical Reviews 2011; 22: 15-29

Antibody Responses of Monkeys and Rabbits to Streptokinase and Staphylokinase.

In our previous experiments, because of the weakness of antigenicity of streptokinase (STK) and staphylokinase (SAK), the antibody response of rabbits and dogs to these extracellular bacterial substances could not be demonstrated by the measurement of antibody titers in the sera of the immunized animals, although the antibody response of humans to STK was proved by demonstrating the rise of anti-STK titers in the sera of human volunteers treated therapeutically with a commercial STK-preparation (Varidase-Lederle). In our present experiments, however, the antigenicity of these substances was clearly demonstrated by the used of monkeys as the experimental animals, in substitution for rabbits, or by the improvement of the immunizing method in rabbits. In the experiments with monkeys anti-STK and anti-SAK were readily produced in the sera of the immunized animals by the primary injections of STK and SAK respectively without adjuvant. In the case of a primary immunization of rabbits, however, the addition of complete Freund\u27s adjuvant to STK or SAK was necessary for the production of anti-STK or anti-SAK in the sera of the immunized animals. Considerablly high titers of anti-STK or anti-SAK were detected in the sera of rabbits, receiving a booster injection after a long lapse of time following the primary injection of the same antigens. The difference between the antibody response of monkeys and that of rabbits to STK and SAK was discussed in connection with natural infection of streptococci and staphylococci among these animal species

敬語行動と規範意識 : 肥筑方言域における言語行動調査から

国立国語研究所日本語の敬語は社会的規範が常にゆれているために，ことば遣いの乱れが問題になる。この問題には，敬語行動の変化とともに，規範意識の変化も関わっている。どういう場面ではどのような敬語表現を選択・付加すべきかというルールが意識の中に形成されたものを規範意識として，地域社会における敬語行動と規範意識の実態を明らかにした。敬意の高い敬語形式を使うことと，規範意識が厳格であることには相関がある。要因分析の結果，ていねいな敬語行動をする人や，敬語に気を遣う必要があると意識している人は規範意識も厳格であることが分かった。また，高学歴層や第三次産業従事者は敬語の規範意識が厳格であることが分かった。In Japanese, standard as well as dialectal, the usage of honorifics can cause social problems, because speakers\u27 attitudes towards the norms of usage are constantly changing. In this paper, the relationship between speakers\u27 choices of honorific expressions and their consciousness of linguistic norms was investigated in the area of Hichiku dialect by means of an interview survey. It was revealed that those who have a distinct consciousness of honorific usage tend to use honorific expressions of higher politeness than those speakers whose consciousness was not distinct. Also, it was revealed that speakers\u27 educational backgrounds and professions were the principal determinants of norm consciousness.17の書名 : 国立国語研究所研究報告

Neutrophil Depletion Exacerbates Pregnancy Complications, Including Placental Damage, Induced by Silica Nanoparticles in Mice

Recent advances in nanotechnology have led to the development of nanoparticles with innovative functions in various fields. However, the biological effects of nanoparticles—particularly those on the fetus—need to be investigated in detail, because several previous studies have shown that various nanoparticles induce pregnancy complications in mice. In this regard, our previous findings in mice suggested that the increase in peripheral neutrophil count induced by treatment with silica nanoparticles with a diameter of 70 nm (nSP70) may play a role in the associated pregnancy complications. Therefore, here, we sought to define the role of neutrophils in nSP70-induced pregnancy complications. The peripheral neutrophil count in pregnant BALB/c mice at 24 h after treatment with nSP70 was significantly higher than in saline-treated mice. In addition, maternal body weight, uterine weight, and the number of fetuses in nSP70-treated mice pretreated with anti-antibodies, which deplete neutrophils, were significantly lower than those in nSP70-treated mice pretreated with phosphate-buffered saline or isotype-matched control antibodies. Histology revealed that neutrophil depletion increased nSP70-induced placental damage from the decidua through the spongiotrophoblast layer and narrowed spiral arteries in the placentae. In addition, depletion of neutrophils augmented nSP70-induced cytotoxicity to fetal vessels, which were covered with endothelium. The rate of apoptotic cell death was significantly higher in the placentae of anti-nSP70-treated mice than in those from mice pretreated with isotype-matched control antibodies. Therefore, impairment of placental vessels and apoptotic cell death due to nSP70 exposure is exacerbated in the placentae of nSP70-treated mice pretreated with anti-antibodies. Depletion of neutrophils worsens nSP70-induced pregnancy complications in mice; this exacerbation was due to enhanced impairment of placental vessels and increased apoptotic cell death in maternal placentae. Our results provide basic information regarding the mechanism underlying silica-nanoparticle-induced pregnancy complications

Sphingosine-1-phosphate signaling and biological activities in the cardiovascular system

金沢大学医薬保健研究域医学系The plasma lysophospholipid mediator sphingosine-1-phosphate (S1P) is produced exclusively by sphingosine kinase (SPHK) 1 and SPHK2 in vivo, and plays diverse biological and pathophysiological roles by acting largely through three members of the G protein-coupled S1P receptors, S1P1, S1P2 and S1P3. S1P1 expressed on endothelial cells mediates embryonic vascular maturation and maintains vascular integrity by contributing to eNOS activation, inhibiting vascular permeability and inducing endothelial cell chemotaxis via Gi-coupled mechanisms. By contrast, S1P2, is expressed in high levels on vascular smooth muscle cells (VSMCs) and certain types of tumor cells, inhibiting Rac and cell migration via a G12/13-and Rho-dependent mechanism. In rat neointimal VSMCs, S1P1 is upregulated to mediate local production of platelet-derived growth factor, which is a key player in vascular remodeling. S1P3 expressed on endothelial cells also mediates chemotaxis toward S1P and vasorelaxation via NO production in certain vascular bed, playing protective roles for vascular integrity. S1P3 expressed on VSMCs and cardiac sinoatrial node cells mediates vasopressor and negative chronotropic effect, respectively. In addition, S1P3, together with S1P2 and SPHK1, is suggested to play a protective role against acute myocardial ischemia. However, our recent work indicates that overexpressed SPHK1 is involved in cardiomyocyte degeneration and fibrosis in vivo, in part through S1P activation of the S1P3 signaling. We also demonstrated that exogenously administered S1P accelerates neovascularization and blood flow recovery in ischemic limbs, suggesting its usefulness for angiogenic therapy. These results provide evidence for S1P receptor subtype-specific pharmacological intervention as a novel therapeutic approach to cardiovascular diseases and cancer. © 2008 Elsevier B.V. All rights reserved

Sphingosine-1-phosphate signaling in physiology and diseases

Sphingosine-1-phosphate (S1P), which acts as both the extracellular and intracellular messenger, exerts pleiotropic biological activities including regulation of formation of the vasculature, vascular barrier integrity, and lymphocyte trafficking. Many of these S1P actions are mediated by five members of the G protein-coupled S1P receptors (S1P 1-S1P 5) with overlapping but distinct coupling to heterotrimeric G proteins. The biological activities of S1P are based largely on the cellular actions of S1P on migration, adhesion, and proliferation. Notably, S1P often exhibits receptor subtype-specific, bimodal effects in these cellular actions. For example, S1P 1 mediates cell migration toward S1P, that is, chemotaxis, via G i/Rac pathway whereas S1P 2 mediates inhibition of migration toward a chemoattractant, that is, chemorepulsion, via G 12/13/Rho pathway, which induces Rac inhibition. In addition, S1P 1 mediates stimulation of cell proliferation through the G i-mediated signaling pathways including phosphatidylinositol 3-kinase (PI3K)/Akt and ERK whereas S1P 2 mediates inhibition of cell proliferation through mechanisms involving G 12/13/Rho/Rho kinase/PTEN-dependent Akt inhibition. These differential effects of S1P receptor subtypes on migration and proliferation lead to bimodal regulation of various biological responses. An observed biological response is likely determined by an integrated outcome of the counteracting signals input by S1P receptor subtypes. More recent studies identified the new intracellular targets of S1P including the inflammatory signaling molecule TRAF2 and histone deacetylases HDAC1 and HDAC2. These interactions of S1P regulate NF-κB activity and gene expression, respectively. Development of S1P receptor agonists and antagonists with improved receptor subtype-selectivity, inhibitors, or modulators of sphingolipid-metabolizing enzymes, and their optimal drug delivery system provide novel therapeutic tactics. © 2012 International Union of Biochemistry and Molecular Biology, Inc