Effective accumulation of polyion complex micelle to experimental choroidal neovascularization in rats

Exudative age-related macular degeneration, characterized by choroidal neovascularization (CNV), is a major cause of visual loss. In this study, we examined the distribution of the polyion complex (PIC) micelle encapsulating FITC-P(Lys) in blood and in experimental CNV in rats to investigate whether PIC micelle can be used for treatment of CNV. We demonstrate that PIC micelle has long-circulating characteristics, accumulating to the CNV lesions and is retained in the lesion for as long as 168 h after intravenous administration. These results raise the possibility that PIC micelles can be used for achieving effective drug targeting to CNV.     

No influence of tumor growth by intramuscular injection of hepatocyte growth factor plasmid DNA: safety evaluation of therapeutic angiogenesis gene therapy in mice

Recently, a novel therapeutic treatment for ischemic diseases using angiogenic growth factors to augment collateral artery development has been proposed. As intramuscular injection of naked human hepatocyte growth factor (HGF) plasmid DNA induced therapeutic angiogenesis in several animal test subjects, we have started a clinical trial to treat peripheral arterial disease. However, one might assume that over-expression of angiogenic growth factors could enhance tumor growth. To resolve this issue, we examined the over-expression of HGF in tumor bearing mice. Tumors on their backs were prepared with an intradermal inoculation of A431, human epidermoid cancer cells expressing c-Met. These mice were intramuscularly injected with human HGF plasmid or control plasmid into the femoral muscle. Human HGF concentration was increased only in the femoral muscle, but not in blood. Although recombinant HGF stimulated the growth of A431 cells in vitro, temporally and locally HGF elevation in hindlimb had no effect on tumor growth in mice.     

Human chromosome 21q22.2-qter carries a gene(s) responsible for downregulation of mlc2a and PEBP in Down syndrome model mice

Congenital heart disease (CHD) is a major clinical manifestation of Down syndrome (DS). We recently showed that chimeric mice containing a human chromosome 21 (Chr 21) exhibited phenotypic traits of DS, including CHD. Our previous study showed that myosin light chain-2a (mlc2a) expression was reduced in the hearts of chimeric mice and DS patients. We found that phosphatidylethanolamine binding protein (PEBP) was also downregulated in Chr 21 chimeras in this study. As mlc2a is involved in heart morphogenesis, and PEBP controls the proliferation and differentiation of different cell types, these genes are candidates for involvement in DS-CHD. The DS-CHD candidate region has been suggested to span between PFKL and D21S3, which is the STS marker near the ETS2 loci. To identify gene(s) or a gene cluster on Chr 21 responsible for the downregulation of mlc2a and PEBP, we fragmented Chr 21 at the EST2 loci, by telomere-directed chromosome truncation in homologous recombination-proficient chicken DT40 cells. The modified Chr 21 was transferred to mouse ES cells by microcell-mediated chromosome transfer (MMCT), via CHO cells. We used ES cell lines retaining the Chr 21 truncated at the ETS2 locus (Chr 21E) to produce chimeric mice and compared overall protein expression patterns in hearts of the chimeras containing the intact and the fragmented Chr 21 by two-dimensional electrophoresis. While mouse mlc2a and PEBP expression was downregulated in the chimeras containing the intact Chr 21, the expression was not affected in the Chr 21E chimeras. Therefore, we suggest that Chr 21 gene(s) distal from the ETS2 locus reduce mouse mlc2a and PEBP expression in DS model mice and DS. Thus, this chromosome engineering technology is a useful tool for identification or mapping of genes that contribute to the DS phenotypes.     

Enhanced tumor-specific long-term immunity of hemagglutinating [correction of hemaggluttinating] virus of Japan-mediated dendritic cell-tumor fused cell vaccination by coadministration with CpG oligodeoxynucleotides

Immunization with dendritic cells (DCs) using various Ag-loading approaches has shown promising results in tumor-specific immunotherapy and immunoprevention. Fused cells (FCs) that are generated from DCs and tumor cells are one of effective cancer vaccines because both known and unknown tumor Ags are presented on the FCs and recognized by T cells. In this study, we attempted to augment antitumor immunity by the combination of DC-tumor FC vaccination with immunostimulatory oligodeoxynucleotides containing CpG motif (CpG ODN). Murine DCs were fused with syngeneic tumor cells ex vivo using inactivated hemagglutinating virus of Japan (Sendai virus). Mice were intradermally (i.d.) immunized with FCs and/or CpG ODN. Coadministration of CpG ODN enhanced the phenotypical maturation of FCs and unfused DCs, and the production of Th1 cytokines, such as IFN-gamma and IL-12, leading to the induction of tumor-specific CTLs without falling into T cell anergy. In addition, immunization with FCs + CpG ODN provided significant protection against lethal s.c. tumor challenge and spontaneous lung metastasis compared with that with either FCs or CpG ODN alone. Furthermore, among mice that rejected tumor challenge, the mice immunized with FCs + CpG ODN, but not the mice immunized with FCs or CpG ODN alone, completely rejected tumor rechallenge, indicating that CpG ODN provided long-term maintenance of tumor-specific immunity induced by FCs. Thus, the combination of DC-tumor FCs and CpG ODN is an effective and feasible cancer vaccine to prevent the generation and recurrence of cancers.     

Plasmacytoid dendritic cells regulate Th cell responses through OX40 ligand and type I IFNs

Dendritic cells (DCs) show a functional plasticity in determining Th responses depending on their maturational stage or on maturational signals delivered to the DCs. Human plasmacytoid DCs (PDCs) can induce either Th1- or Th2-type immune responses upon exposure to viruses or IL-3, respectively. In this study we have investigated the Th-polarizing capacity of PDCs after short (24-h) or long (72-h) culture with stimuli and have assessed the expression and function of OX40 ligand (OX40L) in PDC-mediated Th polarization in addition to type I IFN-dependent responses. IL-3-treated PDCs expressed OX40L, but produced almost no IFN-alpha in response to T cell stimulation (CD40 ligand or T cell interaction), resulting in the preferential priming of Th2 cells through OX40L-dependent mechanisms. Meanwhile, PDCs were rapidly endowed by viral infection (Sendai virus) with a high potency to develop IFN-gamma-producing Th cells depending on their capacity to residually produce IFN-alpha. Although Sendai virus-stimulated PDCs simultaneously expressed OX40L in their maturational process, the Th1-inducing effect of endogenous type I IFNs may overcome and thus conceal the OX40L-dependent Th2 responses. However, during maturation in response to Sendai virus over the longer 72-h period, the expression level of OX40L was up-regulated, whereas the residual IFN-alpha-producing ability was down-regulated, and consequently, the PDCs with prolonged Sendai virus stimulation induced Th2 responses to some extent. Thus, PDCs have the distinct means to dictate an appropriate response to environmental stimuli.     

Development of saxitoxin-conjugated affinity gels

Saxitoxin (STX) and its analogues accumulated in bivalves cause food poisoning through the blockade of sodium channels in the nervous system. In the current studies, STX-conjugated agarose gels as affinity chromatography reagents were prepared for investigation of the fate of the toxins in natural environments and in the human body. A carboxyl moiety was introduced through positions C11 and C13 to leave the most characteristic part of the molecule intact. Two types of synthesized derivatives, 11-(2-carboxyethylthio)saxitoxin and 13-O-hemisuccinyldecarbamoylsaxitoxin, were successfully conjugated to Sepharose 4B in high yield. Affinity gels containing 500 nmol of STX or decarbamoylsaxitoxin per milliliter of gel were accomplished by masking the residual amino groups by acetylation. Finally, the STX-conjugated affinity gel was effective for concentrating STX-binding proteins from pufferfish and bullfrog plasma.     

Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine,N,N′-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N′-diacetylchitobiose [(GlcNAc)₂] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.     

Regeneration of the intestinal epithelia: Regulation of bone marrow-derived epithelial cell differentiation towards secretory lineage cells

The intestinal epithelia consists of four lineages of differentiated cells, all of which arise from stem cells residing in the intestinal crypt. For proper regeneration from epithelial damage, both expansion of the epithelial cell number and appropriate regulation of lineage differentiation from the remaining stem cells are thought to be required. In a series of studies, we have shown that bone-marrow derived cells could promote the regeneration of damaged epithelia in the human intestinal tract. Donor-derived epithelial cells substantially repopulated the gastrointestinal tract of bone-marrow transplant recipients during epithelial regeneration after graft-versus-host disease. Furthermore, precise analysis of epithelial cell lineages revealed that during epithelial regeneration, secretory lineage epithelial cells that originated from bone-marrow significantly increased in number. These findings may lead to a novel therapy to repair damaged intestinal epithelia using bone marrow cells, and provide an alternative therapy for refractory inflammatory bowel diseases.     

Identification of multipotent progenitors in the embryonic mouse kidney by a novel colony-forming assay

Renal stem or progenitor cells with a multilineage differentiation potential remain to be isolated, and the differentiation mechanism of these cell types in kidney development or regeneration processes is unknown. In an attempt to resolve this issue, we set up an in vitro culture system using NIH3T3 cells stably expressing Wnt4 (3T3Wnt4) as a feeder layer, in which a single renal progenitor in the metanephric mesenchyme forms colonies consisting of several types of epithelial cells that exist in glomeruli and renal tubules. We found that only cells strongly expressing Sall1 (Sall1-GFP(high) cells), a zinc-finger nuclear factor essential for kidney development, form colonies, and that they reconstitute a three-dimensional kidney structure in an organ culture setting. We also found that Rac- and JNK-dependent planar cell polarity (PCP) pathways downstream of Wnt4 positively regulate the colony size, and that the JNK pathway is also involved in mesenchymal-to-epithelial transformation of colony-forming progenitors. Thus our colony-forming assay, which identifies multipotent progenitors in the embryonic mouse kidney, can be used for examining mechanisms of renal progenitor differentiation.