Biomedical subjects

Tadanori Mayumi

Publications and source records attributed to Tadanori Mayumi.

At least 19 recordsLinked to original sources

PubMed2006

A strategy for efficient cross-presentation of CTL-epitope peptides leading to enhanced induction of in vivo tumor immunity.

The activation of antitumor cytotoxic T-lymphocytes (CTLs) depends on how efficiently the relevant tumor antigen peptides are delivered into the major histocompatibility complex (MHC) class I presentation pathway in antigen presenting cells (APCs). An elegant approach to promote the peptide-MHC class I association has been described for enhanced peptide transportation into the endoplasmic reticulum (ER) by adding an ER insertion signal sequence (Eriss). Nevertheless, this approach does not appear potent enough to induce in vivo tumor protective immunity. Herein, we present a novel peptide-vaccine strategy based on the combined utilization of Eriss and fusogenic liposomes (FLs) capable of directly introducing encapsulated CTL-epitope peptides into the MHC class I pathway of APCs. APCs pulsed with free peptides, FL-encapsulated peptides, or FL-encapsulated Eriss-conjugated peptides exhibited comparable levels of antigen-presenting activity at early phases after pulsing. Interestingly, whereas in the first two methods the APC ability began to decline 40 to 60 h after pulsing, FL-encapsulated Eriss(+) peptides allowed APCs to retain peptide-presentation activity for at least 140 h. This advantage of FL-encapsulated Eriss(+) peptides correlated with the induction of more potent antitumor immunity compared with soluble Eriss(+) or Eriss(-) peptides or FL-encapsulated Eriss(-) peptides when they were administered in vivo. Thus, Eriss-conjugated CTL-epitope peptides encapsulated in FLs provide a highly efficient tumor-vaccine to enhance the induction of in vivo tumor immunity.

Amino Acid Sequence↗

PubMed2006

A novel method for construction of gene fragment library to searching epitopes.

Identification of the epitope sequence or the functional domain of proteins is a laborious process but a necessary one for biochemical and immunological research. To achieve intensive and effective screening of these functional peptides in various molecules, we established a novel screening method using a phage library system that displays various lengths and parts of peptides derived from target protein. Applying this library for epitope mapping, epitope peptide was more efficiently identified from gene fragment library than conventional random peptide library. Our system may be a most powerful method for identifying functional peptides.

Amino Acid Sequence↗

PubMed2006

Peroxisome proliferator-activated receptor gamma-mediated regulation of neural stem cell proliferation and differentiation.

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays an important role in insulin sensitivity, tissue homeostasis, and regulating cellular functions. We found high-level expression of PPARgamma in embryo mouse brain and neural stem cells (NSCs), in contrast to extremely low levels in adult mouse brain. Here, we show that PPARgamma mediates the proliferation and differentiation of murine NSCs via up-regulation of the epidermal growth factor receptor and activation of the ERK pathway. Cell growth rates of NSCs prepared from heterozygous PPARgamma-deficient mouse brains, PPARgamma-RNA-silenced NSCs, and PPARgamma dominant-negative NSCs were significantly decreased compared with those of wild-type NSCs. Physiological concentrations of PPARgamma agonists, rosiglitazone and pioglitazone, stimulated NSC growth, whereas antagonists caused cell death in a concentration-dependent manner via activation of the caspase cascade. The stimulation of cell growth by PPARgamma was associated with a rapid activation of the ERK pathway by phosphorylation and up-regulation of epidermal growth factor receptor and cyclin B protein levels. In contrast, activation of PPARgamma by agonists inhibited the differentiation of NSCs into neurons. The inhibition of differentiation was associated with an activation of STAT3. These data indicate that PPARgamma regulates the development of the central nervous system during early embryogenesis via control of NSC proliferation.

Animals↗

PubMed2006

Bastadin 6, a spongean brominated tyrosine derivative, inhibits tumor angiogenesis by inducing selective apoptosis to endothelial cells.

Bastadin 6, a macrocyclic tetramer of a brominated tyrosine derivative, was isolated from a marine sponge and its anti-angiogenic activity was evaluated. Bastadin 6 was found to inhibit vascular endothelial growth factor (VEGF)- or basic fibroblast growth factor (bFGF)-dependent proliferation (IC50=0.052 micromol/l) of human umbilical vein endothelial cells (HUVECs) 20- to 100-fold selectively in comparison with normal fibroblast (3Y1) or several tumor cells (KB3-1, K562 and Neuro2A). Bastadin 6 also inhibited VEGF- or bFGF-induced tubular formation (0.1 micromol/l, 6 h treatment) and VEGF-induced migration (1 micromol/l, 4 h treatment) of HUVECs. Moreover, bastadin 6 almost completely blocked VEGF- or bFGF-induced in vivo neovascularization in the mice corneal assay and suppressed growth of s.c. inoculated A431 solid tumor in nude mice (100 mg/kg, i.p.). Bastadin 6 induced cell death of HUVECs with an apoptotic phenotype, whereas it showed no effect on the VEGF-induced auto-phosphorylation of VEGF receptors Flt-1 and KDR/Flk-1. These results suggest that the anti-angiogenic effect of bastadin 6 is closely related to selective induction activity of apoptosis against endothelial cells.

Angiogenesis Inhibitors↗

PubMed2006

Vaccine efficacy of fusogenic liposomes containing tumor cell-lysate against murine B16BL6 melanoma.

Recent advances in tumor immunology have facilitated the development of cancer immunotherapy targeting tumor-associated antigens (TAAs). However, because TAAs were identified in only a few types of human cancer, novel vaccine strategies that utilize tumor cell-lysate (TCL), including unidentified TAAs as an antigen source, are needed. Herein, we describe the utility of fusogenic liposomes (FLs) as TCL-delivery carriers for both ex vivo dendritic cell-based vaccination and in vivo direct immunization in the murine B16BL6 melanoma model. As a result, both in vivo direct immunization and ex vivo immunization induced anti-B16 melanoma prophylactic effects. Ex vivo dendritic cell (DC)-mediated vaccination strategy exert more potent anti-tumor effect than direct immunization. Our results suggest that this flexible system is a promising approach for the development of versatile cancer immunotherapy regimes.

Animals↗

PubMed2006

Non-methylated CpG motif packaged into fusogenic liposomes enhance antigen-specific immunity in mice.

DNA rich in non-methylated CG motifs (CpGs) enhances induction of immune responses against co-administered antigen encoding genes. CpGs are therefore among the promising adjuvants known to date. However, naked plasmid DNA, even which contains CpG motifs, are taken up by antigen presenting cells via the endocytosis pathway. Endocytosed DNAs are thus degraded and their gene expression levels are inefficient. In this context, an effective plasmid delivery carrier is required for DNA vaccine development. We show in the present study that packaging plasmids containing CpGs into fusogenic liposomes (FL) derived from conventional liposomes and Sendai virus-derived active accessory proteins is an attractive method for enhancing the efficacy of a DNA vaccine. These CpG-enhanced plasmids (possessing 16 CpG repeats) that were packaged into FL, enhanced ovalbumin (OVA)-specific T cell proliferation and cytotoxic T cell activity after immunization. In fact, vaccination with CpG enhanced plasmid-loaded FL induced effective prophylactic effects compared with 13 repeats CpG containing plasmid in a tumor challenge experiment. Thus, the development of a CpG-enhanced DNA-FL genetic immunization system represents a promising tool for developing candidate vaccines against some of the more difficult infectious, parasitic, and oncologic disease targets.

Animals↗

PubMed2005

Design and synthesis of a Tat-related gene transporter: a tool for carrying the adenovirus vector into cells.

A Tat-related peptide, acetyl-Gly-Arg-Arg-Arg-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Gly-Cys amide, designed to transport an Adenovirus vector (Ad) into cells, was synthesized. The synthetic peptide was conjugated to Ad, which potentially can act as an efficient carrier of heterologous genes into cells. The Tat-related peptide was synthesized using the solid phase method and then was coupled to the heterofunctional cross-linking reagent, 6-maleimidohexanoic acid N-hydroxysuccinimide ester. The resulting peptide-succinimidohexanoic acid N-hydroxysuccinimide ester was conjugated to Ad containing the luciferase gene. B16BL6 cells infected with the peptide-conjugated Ad luciferase gene construct exhibit a 50-fold greater luciferase activity than B16BL6 cells infected with wild-type Ad containing the luciferase gene.

Adenoviridae↗

PubMed2005

Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release.

Therapeutic agents based on DNA or RNA oligonucleotides (e.g., antisense DNA oligonucleotide, small interfering RNA) require a regulation of their kinetics in cytoplasm to maintain an optimal concentration during the treatment period. In this respect, delivery of functional nanoparticles containing these drugs into cytoplasm has been thought to have a potential for the cytosolic controlled gene release. In this study, we establish a protocol for the encapsulation of nanoparticles into liposome, which is further fused with ultra violet-inactivated Sendai virus to compose fusogenic liposomes. When nanoparticles were encapsulated in conventional liposomes, endocytosis-mediated uptake of nanoparticles was observed. In contrast, numerous amounts of nanoparticles were delivered into the cytoplasm without any cytotoxicity when the particles were encapsulated in fusogenic liposomes. Additionally, fusogenic liposome showed a high ability to deliver nanoparticles containing DNA oligonucleotides into cytoplasm. These results indicate that this combinatorial nanotechnology using fusogenic liposome and nanoparticle is a valuable system for regulating the intracellular pharmacokinetics of gene-based drugs.

Animals↗

PubMed2005

RNA interference of PPARgamma using fiber-modified adenovirus vector efficiently suppresses preadipocyte-to-adipocyte differentiation in 3T3-L1 cells.

The peroxisome proliferator-activated receptor (PPAR) gamma is regarded as a "master regulator" of adipocyte differentiation and is abundantly expressed in adipose. To understand the biological role of PPARgamma in adipose, RNA interference (RNAi) of PPARgamma should be a powerful tool. 3T3-L1 cell line serves an excellent model to investigate the mechanism of preadipocyte-to-adipocyte differentiation. However, this cell line is difficult to transfect by plasmid vectors and viral vectors. We optimized the transduction of both 3T3-L1 preadipocytes and adipocytes by means of fiber-modified adenovirus (Ad) vectors. Among the various vectors tested, polylysine modification of the C-terminal of the fiber knob most markedly improved the transduction efficiency in both 3T3-L1 preadipocytes and adipocytes. Then, we examined whether fiber-modified Ad vectors with polylysine peptides expressing the small interfering RNA (siRNA) for PPARgamma inhibit the differentiation of 3T3-L1 preadipocytes into adipocytes. Oil red O staining and measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity indicated that the vectors effectively suppressed the differentiation of 3T3-L1 preadipocytes to adipocytes. These results suggested that the combination of fiber-modified Ad vectors containing polylysine peptides and RNAi is an effective tool for the study of the biological and physiological mechanism of adipogenesis in adiposity and diabetes using 3T3-L1 models. Ad vector-mediated RNAi for PPARgamma should also be useful to clarify the biological role of the PPARgamma pathway in various tissues in addition to adipose and for therapeutic application to a variety of diseases, including adiposity and diabetes.

3T3-L1 Cells↗

PubMed2005

A single intratumoral injection of a fiber-mutant adenoviral vector encoding interleukin 12 induces remarkable anti-tumor and anti-metastatic activity in mice with Meth-A fibrosarcoma.

Cytokine-encoding viral vectors are considered to be promising in cancer gene immunotherapy. Interleukin 12 (IL-12) has been used widely for anti-tumor treatment, but the administration route and tumor characteristics strongly influence therapeutic efficiency. Meth-A fibrosarcoma has been demonstrated to be insensitive to IL-12 treatment via systemic administration. In the present study, we developed an IL-12-encoding fiber-mutant adenoviral vector (AdRGD-IL-12) that showed enhanced gene transfection efficiency in Meth-A tumor cells, and the production of IL-12 p70 in the culture supernatant from transfected cells was confirmed by ELISA. In therapeutic experiments, a single low-dose (2 x 10(7) plaque-forming units) intratumoral injection of AdRGD-IL-12 elicited pronounced anti-tumor activity and notably prolonged the survival of Meth-A fibrosarcoma-bearing mice. Immunohistochemical staining revealed that the IL-12 vector induced the accumulation of T cells in tumor tissue. Furthermore, intratumoral administration of the vector induced an anti-metastasis effect as well as long-term specific immunity against syngeneic tumor challenge.

Adenoviridae↗

PubMed2005

Nuclear receptors as targets for drug development: the role of nuclear receptors during neural stem cell proliferation and differentiation.

The fate of stem cells, such as neural stem cells and hematopoietic stem cells, depends on strictly regulated signaling events including activation of nuclear receptors, resulting in subsequent gene induction. Recently, we demonstrated that PPARgamma, a ligand-activated nuclear receptor, plays an important role in regulating the proliferation and differentiation of murine neural stem cell (NSC). NSC prepared from heterozygous PPARgamma-deficient mouse exhibited a slower growth rate compared with that of wild-type mouse, which was also demonstrated in PPARgamma-knockdown NSC that was generated by the lentiviral-vector-mediated RNA interference approach. These studies have important implications for understanding central nervous system functions and developing a therapy for neurodegenerative disorders. In this review, recent findings on stem cell biology, especially focusing on nuclear receptors in NSCs, including our current study, will be discussed.

Animals↗

PubMed2005

Design and synthesis of a peptide-PEG transporter tool for carrying adenovirus vector into cells.

The adenovirus vector is a promising carrier for the efficient transfer of genes into cells via the coxackie-adenovirus receptor (CAR) and integrins (alphavbeta3 and alphavbeta5). The clinical use of the adenovirus vector remains problematic however. Successful administration of this vector is associated with side effects because antibodies to this vector are commonly found throughout the human body. To make the adenovirus vector practicable for clinical use, it is necessary to design an auxiliary transporter. The present study describes the use of Arg-Gly-Asp(RGD)-related peptide, a peptide that binds to integrins, as an auxiliary transporter to aid efficient transport of adenovirus vector. Furthermore, poly(ethylene glycol) (PEG) was also used as a tool to modify the adenovirus such that the risk of side effects incurred during clinical application was reduced. The present study describes the design, preparation and use of (acetyl-Tyr-Gly-Gly-Arg-Gly-Asp-Thr-Pro-(beta)Ala)(2)Lys-PEG-(beta)Ala-Cys-NH(2)[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC] as an efficient peptide-PEG transporter tool for carrying adenovirus vector into cells. (Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC was coupled with 6-maleimidohexanoic acid N-hydroxysuccinimide ester and the resulting 6-[(Ac-YGGRGDTP(beta)A)(2)K-PEG-(beta)AC-succinimido]hexanoic acid N-hydroxysuccinimide ester reacted with adenovirus. The modified adenovirus with the peptide-PEG hybrid exhibited high gene expression even in a CAR-negative cell line, DC2.4.

Adenoviridae↗

PubMed2005

PEGylated adenovirus vectors containing RGD peptides on the tip of PEG show high transduction efficiency and antibody evasion ability.

BACKGROUND: PEGylation of adenovirus vectors (Ads) is an attractive strategy in gene therapy. Although many types of PEGylated Ad (PEG-Ads), which exhibit antibody evasion activity and long plasma half-life, have been developed, their entry into cells has been prevented by steric hindrance by polyethylene glycol (PEG) chains. Likewise, sufficient gene expression for medical treatment could not be achieved. METHODS: A set of PEG-Ads, which have different PEG modification rates, was constructed, and gene expression was evaluated using A549 cells. A novel PEGylated Ad (RGD-PEG-Ad), which contained RGD (Arg-Gly-Asp) peptides on the tip of PEG, was developed. We evaluated gene expression both in Coxsackie-adenovirus receptor (CAR)-positive as well as -negative cells, and in vivo gene expression was also determined. Furthermore, the antibody evasion ability and the specificity of infection exhibited by this RGD-PEG-Ad were also evaluated. RESULTS: Whereas PEG-Ads decreased gene expression in CAR-positive cells, RGD-PEG-Ad enhanced gene expression notably, to a level about 200-fold higher than that of PEG-Ads. Moreover, gene expression of RGD-PEG-Ad was almost equal to that of Ad-RGD, which contains an RGD-motif in the fiber and exhibits among the highest gene expression of CAR-positive and -negative cells. Furthermore, although Ad-RGD gene expression decreased remarkably in the presence of anti-Ad antiserum, RGD-PEG-Ad maintained its activity against antibodies. In vivo experiments also demonstrated that the modification of Ads with RGD-PEG induced efficient gene expression. CONCLUSIONS: In the present study, we demonstrated that a new strategy, which combined integrin-targeting the RGD peptide on the tip of PEG and modified the Ad using this material, could enhance gene expression in both CAR-positive and -negative cells. At the same time, this novel PEGylated Ad maintained strong protective activity against antibodies. This strategy could also be easily modified for developing other vectors using other targeting molecules.

Adenoviridae↗

PubMed2005

Immunological properties and vaccine efficacy of murine dendritic cells simultaneously expressing melanoma-associated antigen and interleukin-12.

Interleukin (IL)-12 is a key factor for inducing cellular immune responses, which play a central role in the eradication of cancer. In the present study, in order to create a dendritic cell (DC)-based vaccine capable of positively skewing immune response toward a cellular immunity-dominant state, we analyzed immunological characteristics and vaccine efficacy of DCs cotransduced with melanoma-associated antigen (gp100) and IL-12 gene (gp100+IL12/DCs) by using RGD fiber-mutant adenovirus vector (AdRGD), which enables highly efficient gene transduction into DCs. gp100+IL12/DCs could simultaneously express cytoplasmic gp100 and secretory IL-12 at levels comparable to DCs transduced with each gene alone. In comparison with DCs transduced with gp100 alone (gp100/DCs), upregulation of major histocompatibility complex class I, CD40, and CD86 molecules on the cell surface and more potent T-cell-stimulating ability for proliferation and interferon-gamma secretion were observed as characteristic changes in gp100+IL12/DCs. In addition, administration of gp100+IL12/DCs, which were prepared by a relatively low dose of AdRGD-IL12, could induce more potent tumor-specific cellular immunity in the murine B16BL6 melanoma model than vaccination with gp100/DCs. However, antitumor effect and B16BL6-specific cytotoxic T-lymphocyte activity in mice vaccinated with gp100+IL12/DCs diminished with increasing AdRGD-IL12 dose during gene transduction, and paralleled the decrease in presentation levels via MHC class I molecules for antigen transduced with another AdRGD. Collectively, our results suggested that optimization of combined vector dose was required for development of a more efficacious DC-based vaccine for cancer immunotherapy, which relied on genetic engineering to simultaneously express tumor-associated antigen and IL-12.

Adenoviridae↗

PubMed2005

Transcriptional targeting of RGD fiber-mutant adenovirus vectors can improve the safety of suicide gene therapy for murine melanoma.

Since RGD fiber-mutant adenovirus vector (AdRGD), which contains an alphav-integrin tropism, is highly efficient in gene transduction to melanoma, the AdRGD-mediated herpes simplex virus thymidine kinase (HSVtk)/ganciclovir (GCV) system is an attractive approach for melanoma treatment. However, the intratumoral injection of AdRGD causes limited transgene expression in healthy normal tissue, due to unwanted vector spread. Herein, we describe our attempt to overcome this limitation related to the safety of HSVtk/GCV treatment by using AdRGD carrying either melanoma-specific tyrosinase (Tyr) promoter or tumor-specific telomerase reverse transcriptase (TERT) promoter instead of universal cytomegalovirus promoter. Our in vitro study revealed that Tyr promoter-regulated AdRGD exhibited high transgene expression specificity for melanoma cells, and that TERT promoter-regulated AdRGD could induce efficient gene expression in tumor cells, but was relatively quiescent in normal cells. Anti-B16BL6 melanoma effects in mice injected intratumorally with AdRGD-Tyr/HSVtk or AdRGD-TERT/HSVtk, after which GCV was injected intraperitoneally for 10 days, were comparable to those in mice injected with AdRGD-CMV/HSVtk at 10 times less vector dosage. On the other hand, AdRGD-Tyr/HSVtk and AdRGD-TERT/HSVtk did not induce severe adverse effects even when they were intravenously injected into mice at 10(9) plaque-forming units (PFU), whereas mice injected with AdRGD-CMV/HSVtk at 10(8) PFU exhibited body weight reduction and serum level increase of biochemical enzymes for hepatotoxicity. These results indicate that AdRGD combined with transcriptional regulation using Tyr or TERT promoter is a potentially useful and safe vector system for suicide gene therapy for melanoma.

Adenoviridae↗

PubMed2005

Anti-tumor responses induced by chemokine CCL19 transfected into an ovarian carcinoma model via fiber-mutant adenovirus vector.

Considerable attention has recently been paid to the application of chemokines to cancer immunotherapy because of their chemotactic affinity for a variety of immune cells and because several chemokines are strongly angiostatic. In the present study, the recombinant adenovirus vectors encoding chemokine CCL19 or XCL1 in an E1 cassette (AdRGD-mCCL19 and AdRGD-mXCL1) were developed. The constructed fiber-mutant adenovirus vector, which contained the integrin-targeting Arg-Gly-Asp (RGD) sequence in the fiber knob, notably enhanced the transfection efficiency to OV-HM ovarian carcinoma cells compared to that induced by conventional adenovirus vector. The results of an in vitro chemotaxis assay for chemokine-encoding vector demonstrated that both AdRGD-mCCL19 and AdRGD-mXCL1 could induce the migration of cells expressing specific chemokine receptors. Of the two chemokine-encoding vectors evaluated in vivo, AdRGD-mCCL19 showed significant tumor-suppressive activity in B6C3F1 mice via transduction into OV-HM cells, whereas XCL1 did not exhibit any notable anti-tumor effects, suggesting that CCL19 may be a candidate for cancer immunotherapy.

Adenoviridae↗

PubMed2005

Fusogenic liposome can be used as an effective vaccine carrier for peptide vaccination to induce cytotoxic T lymphocyte (CTL) response.

We reported previously that fusogenic liposome (FL) introduced antigen protein encapsulated in the liposome directly into the cytoplasm of the antigen presenting cells, and that it induced immune responses. In the present study, we encapsulated TAX38-46, an HTLV-I derived protein and an antigen peptide model, into FL. The ability to induce effective cytotoxic T lymphocytes (CTL) responses in immunized mice was evaluated. Results showed FL could induce CTL response effectively and suggested that FL is a potential peptide vaccine carrier.

Animals↗

PubMed2004

Functionalization of tumor necrosis factor-alpha using phage display technique and PEGylation improves its antitumor therapeutic window.

PURPOSE: In this study, the optimization of antitumor therapy with tumor necrosis factor-alpha (TNF-alpha) was attempted. EXPERIMENTAL DESIGN: Using the phage display technique, we created a lysine-deficient mutant TNF-alpha (mTNF-K90R). This mutant had higher affinities to both TNF receptors, despite reports that certain lysine residues play important roles in trimer formation and receptor binding. RESULTS: The mTNF-K90R showed an in vivo therapeutic window that was 13-fold higher than that of the wild-type TNF-alpha (wTNF-alpha). This was due to the synergistic effect of its 6-fold stronger in vitro bioactivity and its 2-fold longer plasma half-life derived from its surface negative potential. The reason why the mTNF-K90R showed a higher bioactivity was understood by a molecular modeling analysis of the complex between the wTNF-alpha and TNF receptor-I. The mTNF-K90R, which was site-specifically mono-PEGylated at the NH2 terminus (sp-PEG-mTNF-K90R), had a higher in vitro bioactivity and considerably longer plasma half-life than the wTNF-alpha, whereas the randomly mono-PEGylated wTNF-alpha had 6% of the bioactivity of the wTNF-alpha. With regard to effectiveness and safety, the in vivo antitumor therapeutic window of the sp-PEG-mTNF-K90R was 60-fold wider than that of the wTNF-alpha. CONCLUSIONS: These results indicated that this functionalized TNF-alpha may be useful not only as an antitumor agent but also as a selective enhancer of vascular permeability in tumors for improving antitumor chemotherapy.

Animals↗