Transcription factor Spi-B-dependent and -independent pathways for the development of Peyer's patch M cells.

Although many of the biological features of microfold cells (M cells) have been known for many years, the molecular mechanisms of M-cell development and antigen recognition have remained unclear. Here, we report that Umod is a novel M-cell-specific gene, the translation products of which might contribute to the uptake function of M cells. Transcription factor Spi-B was also specifically expressed in M cells among non-hematopoietic lineages. Spi-B-deficient mice showed reduced expression of most, but not all, other M-cell-specific genes and M-cell surface markers. Whereas uptake of Salmonella Typhimurium via M cells was obviously reduced in Spi-B-deficient mice, the abundance of intratissue cohabiting bacteria was comparable between wild-type and Spi-B-deficient mice. These data indicate that there is a small M-cell population with developmental regulation that is Spi-B independent; however, Spi-B is probably a candidate master regulator of M-cell functional maturation and development by another pathway.

A marvel of mucosal T cells and secretory antibodies for the creation of first lines of defense.

The mucosal immune system acts as a first line of defense against bacterial and viral infections while also playing a crucial role in the establishment and maintenance of mucosal homeostasis between the host and the outside environment. In addition to epithelial cells and antigen-presenting cells (dendritic cells and macrophages), B and T lymphocytes form a dynamic mucosal network for the induction and regulation of secretory IgA (S-IgA) and cytotoxic T lymphocyte (CTL) responses. This review seeks to shed light on the pathways of induction and regulation of these responses and to elucidate the role they simultaneously play in fending off pathogen invasion and maintaining mucosal homeostasis.

Pharmacotherapy by intracellular delivery of drugs using fusogenic liposomes: application to vaccine development.

We prepared fusogenic liposomes by fusing conventional liposomes with an ultra-violet inactivated Sendai virus. Fusogenic liposomes can deliver encapsulated contents into the cytoplasm directly in a Sendai virus fusion-dependent manner. Based on the high delivery rates into the cytoplasm, we originally planned to apply the fusogenic liposomes to cancer chemotherapy and gene therapy. We have recently also examined the use of fusogenic liposomes as an antigen delivery vehicle. In terms of vaccine development, cytoplasmic delivery is crucial for the induction of the cytotoxic T lymphocyte (CTL) responses that play a pivotal role against infectious diseases and cancer. In this context, our recent studies suggested that fusogenic liposomes could deliver encapsulated antigens into the cytoplasm and induce MHC class I-restricted, antigen-specific CTL responses. In addition, fusogenic liposomes are also effective as a mucosal vaccine carrier. In this review, we present the feasibility of fusogenic liposomes as a versatile and effective antigen delivery system.

Sendai virus fusion protein mediates simultaneous induction of MHC class I/II-dependent mucosal and systemic immune responses via the nasopharyngeal-associated lymphoreticular tissue immune system.

Nasal administration of Ags using a novel hybrid Ag delivery vehicle composed of envelope glycoproteins of Sendai virus on the surface of liposome membranes (fusogenic liposome) efficiently delivered Ags to Ag-sampling M cells in nasopharyngeal-associated lymphoreticular tissue. Additionally, fusogenic liposomes also effectively delivered the Ags into epithelial cells and macrophages in nasopharyngeal-associated lymphoreticular tissue and nasal passages. In vitro Ag presentation assays clearly showed that fusogenic liposomes effectively presented encapsulated Ags via the MHC class II-dependent pathway of epithelial cells as well as macrophages. Fusogenic liposomes also have an adjuvant activity against mucosal epithelial cells to enhance MHC class II expression. According to these high delivery and adjuvant activities of fusogenic liposomes, nasal immunization with OVA-encapsulated fusogenic liposomes induced high levels of OVA-specific CD4(+) Th1 and Th2 cell responses. Furthermore, Ag-specific CTL responses and Ab productions were also elicited at both mucosal and systemic sites by nasal immunization with Ag-encapsulated fusogenic liposomes. These results indicate that fusogenic liposome is a versatile and effective system for the stimulation of Ag-specific immune responses at both mucosal and systemic compartments.

[Application of novel drug delivery system, fusogenic liposome, for cancer therapy].

Over the past decade, many studies concerning novel anti-cancer therapies have been reported. It has been occasionally noted that a powerful anti-cancer drug, especially one whose target is the cytoplasm or cell nucleus, does not work due to the low permeability across a plasma membrane, degradation by lysosomal enzymes through an endocytosis-dependent pathway, and other reasons. Thus, several approaches using drug delivery systems (DDS) are focused on overcoming these difficulties, eventually leading to the induction of maximal ability of anti-cancer drug. In this respect, we have developed a new paradigm for cancer therapy using a novel drug delivery system, fusogenic liposome. Fusogenic liposomes are composed of the ultraviolet-inactivated Sendai virus and conventional liposomes. Fusogenic liposomes effectively and directly deliver their encapsulated contents into the cytoplasm using a fusion mechanism of the Sendai virus, whereas conventional liposomes are taken up by endocytosis. Thus, fusogenic liposome is a good candidate as a vehicle to deliver drugs into the cytoplasm in an endocytosis-independent manner. In this report, we show the feasibility of fusogenic liposome as a delivery vehicle for anti-cancer drugs using a fragment A of diphtheria toxin as an anti-cancer reagent. We also demonstrate the application of fusogenic liposome for cancer gene therapy and cancer vaccines using a TNF-alpha-expression plasmid and a chicken egg ovalbumin, respectively.

Characterization of mucoadhesive microspheres for the induction of mucosal and systemic immune responses.

In the present study, mucoadhesive polymer-dispersed microspheres (MS) were examined as a potential mucosal vaccine carrier. A major focus of the study was aimed at directly assessing the influence of antigen release and persistence in the mouse small intestine for the induction of mucosal and systemic immune responses. BALB/c mice were immunized with various forms of MS containing chicken egg ovalbumin (OVA) by administration into the duodenum. No detectable anti-OVA immune responses were observed following the administration of OVA alone or that of MS without mucoadhesive polymer (MS-0). MS-10 containing 10% mucoadhesive polymer rapidly released OVA and hardly induced anti-OVA antibody responses in either serum or fecal extracts. In contrast, MS-8 and MS-6 (with 8 and 6% mucoadhesive polymer) showed controlled release of OVA, which elicited strong OVA-specific IgG and IgA responses in serum and fecal extracts, respectively. Additionally, the strongest immune responses were induced in mice immunized with MS-8, which had both the optimal release-profile of OVA and the longest persistence in the small intestine. These findings indicate that antigen movement in the small intestine is an important factor and that appropriate microsphere forms with mucoadhesive polymers might be useful candidates as mucosal vaccine carriers.

Fusogenic liposomes efficiently deliver exogenous antigen through the cytoplasm into the MHC class I processing pathway.

Exogenous soluble proteins enter the endosomal pathway by endocytosis and are presented in association with MHC class II rather than class I. In contrast, the delivery of exogenous protein antigens (Ag) into the cytosol generates MHC class I-restricted cytotoxic T lymphocytes (CTL) responses. Although several immunization approaches, such as the utilization of liposomes, have induced the in vivo priming of MHC class I-restricted CTL responses to protein Ag, it remains unclear whether this priming results from the direct delivery of protein Ag to the cytosol. Here we report that fusogenic liposomes (FL), which are prepared by fusing simple liposomes with Sendai virus particles, can deliver the encapsulated soluble protein directly into the cytosol of cells cultured concurrently and introduce it into the conventional MHC class I Ag presentation pathway. Moreover, a single immunization with ovalbumin (OVA) encapsulated in FL but not in simple liposomes results in the potent priming of OVA-specific CTL. Thus, FL function as an efficient tool for the delivery of CTL vaccines.

Positively charged liposome functions as an efficient immunoadjuvant in inducing cell-mediated immune response to soluble proteins.

In order to design an optimized liposome immunoadjuvant for inducing cell-mediated immune response against soluble proteinaceous antigens, we investigated the effect of liposomal surface charge on the immunoadjuvant action. Positively charged liposomes containing soluble antigens functioned as a more potent inducer of antigen-specific cytotoxic T lymphocyte responses and delayed type hypersensitivity response than negatively charged and neutral liposomes containing the same concentrations of antigens. To clarify the reason of the differential immune response, we examined the delivery of soluble proteins by the liposomes into the cytoplasm of macrophages, using fragment A of diphtheria toxin (DTA) as a marker. We found that positively charged liposomes encapsulating DTA are cytotoxic to macrophages, while empty positively charged liposomes, DTA in negatively charged and neutral liposomes are not. Consistent with this, only macrophages pulsed with OVA in positively charged liposomes could significantly stimulate OVA-specific, class I MHC-restricted T cell hybridoma. These results suggest that the positively charged liposomes can deliver proteinaceous antigens efficiently into the cytoplasm of the macrophages/antigen-presenting cells, where the antigens are processed to be presented by class I MHC molecules to induce the cell-mediated immune response. Possible development of the safe and effective vaccine is discussed.

A novel vaccine delivery system using immunopotentiating fusogenic liposomes.

We previously reported the preparation and characterization of fusogenic liposomes (FLs), which have two highly immunogenic glycoproteins of the Sendai virus on their surface. In this report, we investigated the capacity of FLs to enhance antigen-specific humoral immunity in mice. FLs function as a lymphocyte mitogen with high immunogenicity consistent with viral envelope proteins. Markedly increased levels of anti-ovalbumin (OVA) antibody were detected in serum from mice immunized with OVA encapsulated in FLs compared to sera from mice immunized with free OVA or OVA encapsulated in plain liposomes. An anti-OVA antibody response was not observed in mice immunized with OVA simply mixed with empty FLs. These results indicate that FLs function as a novel immunoadjuvant in inducing antigen-specific antibody production.

Positively charged liposome functions as an efficient immunoadjuvant in inducing immune responses to soluble proteins.

To design an optimum liposome immunoadjuvant for soluble protein antigens, we investigated the relationship between liposomal surface charge and adjuvant action. Positively charged multilamellar vesicles (MLV) were taken up efficiently by macrophages, while negatively charged and neutral MLVs were hardly picked up. Consistent with this, positively charged MLVs containing soluble chicken egg albumin (OVA) functioned as a more potent inducer of antigen-specific cytotoxic T lymphocyte (CTL) responses and antibody production than negatively charged and neutral MLVs containing the same concentrations of antigens. These results indicate that the positive charge on the surface of liposomes represents an important factor for enhancing their immunoadjuvancy in the induction of antigen-specific immune responses.