Delivery of dendritic cells engineered to secrete IFN-alpha into central nervous system tumors enhances the efficacy of peripheral tumor cell vaccines: dependence on apoptotic pathways

We tested whether modulation of the CNS-tumor microenvironment by delivery of IFN-alpha-transduced dendritic cells (DCs: DC-IFN-alpha) would enhance the therapeutic efficacy of peripheral vaccinations with cytokine-gene transduced tumor cells. Mice bearing intracranial GL261 glioma or MCA205 sarcoma received peripheral immunizations with corresponding irradiated tumor cells engineered to express IL-4 or GM-CSFs, respectively, as well as intratumoral delivery of DC-IFN-alpha. This regimen prolonged survival of the animals and induced tumor-specific CTLs that expressed TRAIL, which in concert with perforin and Fas ligand (FasL) was involved in the tumor-specific CTL activity of these cells. The in vivo antitumor activity associated with this approach was abrogated by administration of neutralizing mAbs against TRAIL or FasL and was not observed in perforin-/-, IFN-gamma-/-, or FasL-/- mice. Transduction of the tumor cells with antiapoptotic protein cellular FLIP rendered the gene-modified cells resistant to TRAIL- or FasL-mediated apoptosis and to CTL killing activity in vitro. Furthermore, the combination therapeutic regimen was ineffective in an intracranial cellular FLIP-transduced MCA205 brain tumor model. These results suggest that the combination of intratumoral delivery of DC-IFN-alpha and peripheral immunization with cytokine-gene transduced tumor cells may be an effective therapy for brain tumors that are sensitive to apoptotic signaling pathways.     

Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation

Bacterial DNA contains immunostimulatory CpG motifs that trigger an innate immune response capable of promoting host survival following infectious challenge. Yet CpG-driven immune activation may also have deleterious consequences, ranging from autoimmune disease to death. We find that repetitive elements present at high frequency in mammalian telomeres, but rare in bacteria, down-regulate CpG-induced immune activation. Suppressive activity correlates with the ability of telomeric TTAGGG repeats to form G-tetrads. Colocalization of CpG DNA with Toll-like receptor 9 in endosomal vesicles is disrupted by these repetitive elements, although cellular binding and uptake remain unchanged. These findings are the first to establish that specific host-derived molecules can down-regulate the innate immune response elicited by a TLR ligand.     

A new En face method is useful to quantitate endothelial damage in vivo

Endothelial damage is considered to be an initial change in the atherosclerotic process. However, it has been difficult to detect this initial change in vivo. We established a modified En face immunostaining method that enabled us to obtain clear images of the entire endothelial surface, including at arterial bifurcations, and to quantitate the number of cells of interest in the endothelium. Using this method, we found that treatment with an atherogenic factor, albumin-derived advanced glycosylation end products, for only 2 weeks caused a significant increase in the number of proliferating cell nuclear antigen-positive endothelial cells and the number of macrophages adhering to the endothelium, suggesting that these changes might be relevant to the early events of endothelial dysfunction. In conclusion, the present modified En face immunostaining method may be a promising tool for understanding the pathophysiology of atherosclerosis.     

Association of TAG-1 with Caspr2 is essential for the molecular organization of juxtaparanodal regions of myelinated fibers

Myelination results in a highly segregated distribution of axonal membrane proteins at nodes of Ranvier. Here, we show the role in this process of TAG-1, a glycosyl-phosphatidyl-inositol-anchored cell adhesion molecule. In the absence of TAG-1, axonal Caspr2 did not accumulate at juxtaparanodes, and the normal enrichment of shaker-type K+ channels in these regions was severely disrupted, in the central and peripheral nervous systems. In contrast, the localization of protein 4.1B, an axoplasmic partner of Caspr2, was only moderately altered. TAG-1, which is expressed in both neurons and glia, was able to associate in cis with Caspr2 and in trans with itself. Thus, a tripartite intercellular protein complex, comprised of these two proteins, appears critical for axo-glial contacts at juxtaparanodes. This complex is analogous to that described previously at paranodes, suggesting that similar molecules are crucial for different types of axo-glial interactions.     

Limonene production in tobacco with Perilla limonene synthase cDNA

Limonene synthase (LS) catalyses the stereo-specific cyclization of geranyl diphosphate (GPP) to form a monocyclic monoterpene, limonene. In an attempt to engineer monoterpene biosynthesis, three expression constructs of LS cDNA of Perilla frutescens, which were designed to be localized in either the plastid, the cytosol or the endoplasmic reticulum (ER), were introduced into tobacco in order to examine differences in enzyme activity and the productivity of limonene. High and moderate enzyme activity, respectively, was observed for plastid- and cytosol-localized LS, whereas no enzyme activity was seen for ER-localized LS, suggesting that the plastid is the preferred compartment for LS, while LS may also have an active form in the cytosol. The formation of limonene in vivo was confirmed by gas chromatography-mass spectrometry (GC-MS) in leaf extracts of both plastid- and cytosol-localized LS transgenic plants. The amount of limonene in plastid-localized LS transgenic plants was 143 ng g-1 fresh wt, whereas that in the cytosol-type was 40 ng g-1 fresh wt, and these limonene contents increased by 2.7-fold and 3.0-fold, respectively, with the addition of methyl jasmonate. The headspace analyses showed that the plastid- and the cytosol-localized LS transgenic plants (12 cm high) emitted 390 ng and 515 ng limonene per month, respectively. The possibility of genetically engineering monoterpene production is discussed.     

Roles of hydration water molecules in molecular packing of the killer toxin from Pichia farinosa in its crystalline state investigated by cryogenic X-ray crystallography

The hydration structures around the killer toxin from Pichia farinosa were investigated by cryogenic X-ray crystallography. In particular, those contributing to the molecular association and the crystal contacts were analyzed with respect to the geometry and the networks of hydrogen bonds. The hydration water molecules attached on the surface so as to make up the surface shape in the contact complementary and mediated the intermolecular interactions through the networks of hydrogen bonds. Careful inspection of the contact area led to a proposal as to the molecular association mode of the toxin to determine the biological function in cells. In addition, the water-associated protein-protein interactions were approximated well by a simple theoretical equation on the solvation force expected in confined geometry. The present analysis may provide a way to analyze the crystal contact and molecular recognition in macromolecules in aqueous solution.     

Effect of suppressive DNA on CpG-induced immune activation

Bacterial DNA and synthetic oligodeoxynucleotides (ODN) containing unmethylated CpG motifs stimulate a strong innate immune response. This stimulation can be abrogated by either removing the CpG DNA or adding inhibitory/suppressive motifs. Suppression is dominant over stimulation and is specific for CpG-induced immune responses (having no effect on LPS- or Con A-induced activation). Individual cells noncompetitively internalize both stimulatory and suppressive ODN. Studies using ODN composed of both stimulatory and suppressive motifs indicate that sequence recognition proceeds in a 5'-->3' direction, and that a 5' motif can block recognition of immediately 3' sequences. These findings contribute to our understanding of the immunomodulatory activity of DNA-based products and the rules that govern immune recognition of stimulatory and suppressive motifs.     

M6a acts as a nerve growth factor-gated Ca(2+) channel in neuronal differentiation

To elucidate the function of M6a, which is a neuron-specific membrane glycoprotein of the brain and possesses putative phosphorylation sites for protein kinase C (PKC), we established rat M6a cDNA expression vector-transfected PC12 cells. These transfectants exhibited high susceptibilities to nerve growth factor (NGF) for neuronal differentiation. Interestingly, we found that Ca(2+) influx in these transfectants was significantly augmented by the treatment of NGF, but not epidermal growth factor (EGF), which stimulates PC12 cell growth. NGF-dependent augmentation of Ca(2+) influx was detected within 3h and severely inhibited by EGTA- and PKC-specific inhibitors. Anti-M6 antibody suppressed both NGF-triggered Ca(2+) influx and neuronal differentiation. These results support the idea that M6a implicates in neuronal differentiation as a novel Ca(2+) channel gated selectively by phosphorylation with PKC in the downstream of NGF signaling pathway.