Reversible integration of the dominant negative retinoid receptor gene for ex vivo expansion of hematopoietic stem/progenitor cells

Proliferation of vascular smooth muscle cells (VSMC) contributes to the pathogenesis of atherosclerosis, and glycated serum albumin (GSA, Amadori adduct of albumin) might be a mitogen for VSMC proliferation, which may further be associated with diabetic vascular complications. In this study, we investigated the involvement of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK), and protein kinase C (PKC), in GSA-stimulated mitogenesis, as well as the functional relationship between these factors. VSMC stimulation with GSA resulted in a marked activation of ERK. The MAPK kinase (MEK) inhibitor, PD98059, blocked GSA-stimulated MAPK activation and resulted in an inhibition of GSA-stimulated VSMC proliferation. GSA also increased PKC activity in VSMC in a dose-dependent manner. The inhibition of PKC by the PKC inhibitors, GF109203X and Rottlerin (PKCdelta specific inhibitor), as well as PKC downregulation by phorbol 12-myristate 13-acetate (PMA), inhibited GSA-induced cell proliferation and blocked ERK activation. This indicates that phorbol ester-sensitive PKC isoforms including PKCdelta are involved in MAPK activation. Thus, we show that the MAPK cascade is required for GSA-induced proliferation, and that phorbol ester-sensitive PKC isoforms contribute to cell activation and proliferation in GSA-stimulated VSMC.     

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5)

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.     

Further Studies of the Polysaccharide of Klebsiella pneumoniae Possessing Strong Adjuvanticity: III. Augmentation of the Antibody Response to Subcutaneously Injected Sheep Red Blood Cells by the Adjuvant Polysaccharide

The adjuvant action of the O3 antigen of Klebsiella (KO3) on the antibody response to sheep red blood cells (SRBC) was elucidated by injecting both KO3 and SRBC subcutaneously at the right inguinal region of SMA mice. We demonstrated that KO3 exhibits a novel ability to augment anti-SRBC plaque-forming cell responses in both the local lymph node and the spleen at a relatively late stage of immunization. Escherichia coli lipopolysaccharide, dextran sulfate and concanavalin A showed such an action only minimally. In parallel with the development of the adjuvant action, KO3 definitely activated B cells in the local lymph node polyclonally for either IgM or IgG synthesis, suggesting that the mechanism of the adjuvant action includes direct stimulation of B cells by KO3 at the late stage. Neither increase in trapping of lymphocytes in the local lymph node nor change in tissue distribution of antigen was shown to be primarily involved in the mechanism of the adjuvant action.     

Lignin Induces ES Cells to Differentiate into Neuroectodermal Cells through Mediation of the Wnt Signaling Pathway

Embryonic stem cells (ES cells) are characterized by their pluripotency and infinite proliferation potential. Ever since ES cells were first established in 1981, there have been a growing number of studies aimed at clinical applications of ES cells. In recent years, various types of differentiation inducement systems using ES cells have been established. Further studies have been conducted to utilize differentiation inducement systems in the field of regenerative medicine. For cellular treatments using stem cells including ES cells, differentiation induction should be performed in a sufficient manner to obtain the intended cell lineages. Lignin is a high-molecular amorphous material that forms plants together with cellulose and hemicelluloses, in which phenylpropane fundamental units are complexly condensed. Lignin derivatives have been shown to have several bioactive functions. In spite of these findings, few studies have focused on the effects of lignin on stem cells. Our study aimed to develop a novel technology using lignin to effectively induce ES cells to differentiate into neuroectodermal cells including ocular cells and neural cells. Since lignin can be produced at a relatively low cost in large volumes, its utilization is expected for more convenient differentiation induction technologies and in the field of regenerative medicine in the future.     

Contribution of IL-12/IL-35 Common Subunit p35 to Maintaining the Testicular Immune Privilege

The testis is an organ with immune privilege. The comprehensive blood–testis barrier formed by Sertoli cells protects autoimmunogenic spermatozoa and spermatids from attack by the body’s immune system. The interleukin (IL)-6/IL-12 family cytokines IL-12 (p35/p40), IL-23 (p19/p40), IL-27 (p28/Epstein-Barr virus−induced gene 3 [EBI3]), and IL-35 (p35/EBI3) play critical roles in the regulation of various immune responses, but their roles in testicular immune privilege are not well understood. In the present study, we investigated whether these cytokines are expressed in the testes and whether they function in the testicular immune privilege by using mice deficient in their subunits. Expression of EBI3 was markedly increased at both mRNA and protein levels in the testes of 10- or 12-week-old wild-type mice as compared with levels in 2-week-old mice, whereas the mRNA expression of p40 was markedly decreased and that of p35 was conserved between these two groups. Lack of EBI3, p35, and IL-12 receptor β2 caused enhanced infiltration of lymphocytes into the testicular interstitium, with increased interferon-γ expression in the testes and autoantibody production against mainly acrosomal regions of spermatids. Spermatogenic disturbance was more frequently observed in the seminiferous tubules, especially when surrounded by infiltrating lymphocytes, of these deficient mice than in those of wild-type mice. In particular, p35-deficient mice showed the most severe spermatogenic disturbance. Immunohistochemical analyses revealed that endothelial cells and peritubular cells in the interstitium were highly positive for p35 at both ages, and CD163⁺ resident macrophages positive for p35 and EBI3, possibly producing IL-35, were also detected in the interstitium of 12-week-old mice but not those of 2-week-old mice. These results suggest that p35 helps in maintaining the testicular immune privilege, in part in an IL-35-dependent manner.     

A new model cage for simultaneous measurements of wheel-running and ambulatory activity in the rat

A new type of cage including a running wheel basket and tilting floor was developed. Using this cage it was possible to record simultaneously wheel-running and ambulatory activity of the rat.     

Expression and Characterization of Recombinant,Tetrameric and Enzymatically Active Influenza Neuraminidase for the Setup of an Enzyme-Linked Lectin-Based Assay

Developing a universal influenza vaccine that induces broad spectrum and longer-term immunity has become an important potentially achievable target in influenza vaccine research and development. Hemagglutinin (HA) and neuraminidase (NA) are the two major influenza virus antigens. Although antibody responses against influenza virus are mainly directed toward HA, NA is reported to be more genetically stable; hence NA-based vaccines have the potential to be effective for longer time periods. NA-specific immunity has been shown to limit the spread of influenza virus, thus reducing disease symptoms and providing cross-protection against heterosubtypic viruses in mouse challenge experiments.The production of large quantities of highly pure and stable NA could be beneficial for the development of new antivirals, subunit-based vaccines, and novel diagnostic tools. In this study, recombinant NA (rNA) was produced in mammalian cells at high levels from both swine A/California/07/2009 (H1N1) and avian A/turkey/Turkey/01/2005 (H5N1) influenza viruses. Biochemical, structural, and immunological characterizations revealed that the soluble rNAs produced are tetrameric, enzymatically active and immunogenic, and finally they represent good alternatives to conventionally used sources of NA in the Enzyme-Linked Lectin Assay (ELLA).     

Galectin-9 Is a High Affinity IgE-binding Lectin with Anti-allergic Effect by Blocking IgE-Antigen Complex Formation

Galectin (Gal)-9 was first described as an eosinophil chemoattractant. With the progress in research, Gal-9 has come to be known as a versatile immunomodulator that is involved in various aspects of immune regulations, and the entire picture of the function still remains elusive. To uncover as-yet unknown activity of Gal-9, we have been examining the effect of the protein in various disease animal models. Here we show that Gal-9 attenuated asthmatic reaction in guinea pigs and suppressed passive-cutaneous anaphylaxis in mice. These results indicate the mast cell stabilizing effect of Gal-9. In vitro studies of mast cell degranulation involving RBL-2H3 cells demonstrated that Gal-9 suppressed degranulation from the cells stimulated by IgE plus antigen and that the inhibitory effect was completely abrogated in the presence of lactose, indicating lectin activity of Gal-9 is critical. We found that Gal-9 strongly and specifically bound IgE, which is a heavily glycosylated immunoglobulin, and that the interaction prevented IgE-antigen complex formation, clarifying the mode of action of the anti-degranulation effect. Gal-9 is expressed by several mast cells including mouse mast cell line MC/9. The fact that immunological stimuli of MC/9 cells augmented Gal-9 secretion from the cells implies that Gal-9 is an autocrine regulator of mast cell function to suppress excessive degranulation. Collectively, these findings shed light on a novel function of Gal-9 in mast cells and suggest a beneficial utility of Gal-9 for the treatment of allergic disorders including asthma.