Exosomes are a valuable biomaterial for the development of novel nanocarriers as functionally advanced drug delivery systems. To control and modify the performance of exosomal nanocarriers, we developed hybrid exosomes by fusing their membranes with liposomes using the freeze–thaw method. Exosomes embedded with a specific membrane protein isolated from genetically modified cells were fused with various liposomes, confirming that membrane engineering methods can be combined with genetic modification techniques. Cellular uptake studies performed using the hybrid exosomes revealed that the interactions between the developed exosomes and cells could be modified by changing the lipid composition or the properties of the exogenous lipids. These results suggest that the membrane-engineering approach reported here offers a new strategy for developing rationally designed exosomes as hybrid nanocarriers for use in advanced drug delivery systems.

Nanotechnology is an innovative method of freely controlling nanometre-sized materials. Recent outbreaks of mucosal infectious diseases have increased the demands for development of mucosal vaccines because they induce both systemic and mucosal antigen-specific immune responses. Here we developed an intranasal vaccine-delivery system with a nanometre-sized hydrogel ('nanogel') consisting of a cationic type of cholesteryl-group-bearing pullulan (cCHP). A non-toxic subunit fragment of Clostridium botulinum type-A neurotoxin BoHc/A administered intranasally with cCHP nanogel (cCHP-BoHc/A) continuously adhered to the nasal epithelium and was effectively taken up by mucosal dendritic cells after its release from the cCHP nanogel. Vigorous botulinum-neurotoxin-A-neutralizing serum IgG and secretory IgA antibody responses were induced without co-administration of mucosal adjuvant. Importantly, intranasally administered cCHP-BoHc/A did not accumulate in the olfactory bulbs or brain. Moreover, intranasally immunized tetanus toxoid with cCHP nanogel induced strong tetanus-toxoid-specific systemic and mucosal immune responses. These results indicate that cCHP nanogel can be used as a universal protein-based antigen-delivery vehicle for adjuvant-free intranasal vaccination.

The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Ras (Ha-Ras). The amino acid sequences of the peptides derived from p180 were almost identical to those of human AF-6 that is identified as the fusion partner of the ALL-1 protein.The ALL-1/AF-6 chimeric protein is the critical product of the t (6:11) abnormality associated with some human leukemia. AF-6 has a GLGF/Dlg homology repeat (DHR) motif and shows a high degree of sequence similarity with Drosophila Canoe, which is assumed to function downstream from Notch in a common developmental pathway. The recombinant N-terminal domain of AF-6 and Canoe specifically interacted with GTP␥S⅐GST-HaRas. The known Ras target c-Raf-1 inhibited the interaction of AF-6 with GTP␥S⅐GST-Ha-Ras. These results indicate that AF-6 and Canoe are putative targets for Ras.Ras (Ha-Ras, Ki-Ras, N-Ras) is a signal-transducing guanine nucleotide-binding protein for tyrosine kinase-type receptors such as epidermal growth factor receptors and the Src family, leading to a mitogenic response and differentiation (for reviews, see Refs. 1 and 2). Ras has GDP-bound inactive and GTP-bound active forms, the latter of which makes physical contact with targets. Intensive investigations revealed that the Raf kinase family, consisting of c-Raf-1 (for reviews, see Refs. 3 and 4), A-Raf (5), and B-Raf (6 -9), is one of the direct targets for Ras. The activated Raf phosphorylates MAP 1 kinase kinase and activates it. Consequently the activated MAP kinase kinase activates MAP kinase, leading to the expression of certain genes such as c-fos (for reviews, see Refs. 10 and 11). Several molecules interacting with activated Ras in addition to Raf have been identified in mammals. These include phosphatidylinositol-3-OH kinase (12), Ral GDS (13,14), and Rin1 (15). On the basis of these observations, a variety of Ras targets may account for the pleiotropic functions of Ras. To understand the molecular mechanism of pleiotropic functions of Ras, it is essential to identify novel targets for Ras.In the present study, we discovered and partially purified another putative target for Ras with a molecular mass of about 180 kDa (p180) by use of GST-Ha-Ras affinity column chromatography and identified it as AF-6 (16), whose structure resembles that of Drosophila Canoe, which is involved in the Notch signaling pathway (17). S]methionine were purchased from DuPont NEN. A rabbit polyclonal antibody against a 16-mer peptide corresponding to 561-576 aa of human AF-6 (RVEQQPDYRRQESRTQ) was generated and purified. EXPERIMENTAL PROCEDURES Materials and Chemicals-AllPlasmid Construction-Plasmids, pGEX-Ha-Ras, pGEX-R-Ras, pGEX-RalA, and pGEX-RhoA were constructed as described previously (18). To obtain the in vitro translated N-terminal domain of AF-6 and Canoe, pRSET-AF-6 (36 -848 aa), pRSET-AF-6 (36 -206 aa), and pR-SET-Canoe (1-217 aa) were constructed as follows. The 2.4-kilobase cDNA fragment encoding AF-6 (36 -848 aa) was amplified by polymerase chain reaction from human brain Quick clone cDNA (Clontech Laboratories Inc., Palo Alto, CA). For the shorter N-terminal domain of ...

Fibroblastic tumour stroma comprising mesenchymal stem cells (MSCs) and cancer-associated fibroblasts (CAFs) promotes the invasive and metastatic properties of tumour cells. Here we show that activated CD8+ T cell-derived extracellular vesicles (EVs) interrupt fibroblastic stroma-mediated tumour progression. Activated CD8+ T cells from healthy mice transiently release cytotoxic EVs causing marked attenuation of tumour invasion and metastasis by apoptotic depletion of mesenchymal tumour stromal cells. Infiltration of EV-producing CD8+ T cells is observed in neovascular areas with high mesenchymal cell density, and tumour MSC depletion is associated with preferential engulfment of CD8+ T cell EVs in this setting. Thus, CD8+ T cells have the capacity to protect tumour progression by EV-mediated depletion of mesenchymal tumour stromal cells in addition to their conventional direct cytotoxicity against tumour cells.