Biomedical subjects

Yoshinobu Takakura

Publications and source records attributed to Yoshinobu Takakura.

At least 19 recordsLinked to original sources

PubMed2006

DNA and its cationic lipid complexes induce CpG motif-dependent activation of murine dendritic cells.

Unmethylated CpG motifs in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response of antigen-presenting cells (APC). In this study, we investigated the cytokine release from murine dendritic cells (DC) by the addition of various types of DNA in the free or complexed form with cationic lipids. Naked plasmid DNA and Escherichia coli DNA with immunostimulatory unmethylated CpG motifs induced pro-inflammatory cytokine secretion from granulocyte-macrophage colony-stimulating factor (GM-CSF)-cultured bone marrow-derived DC and the DC cell-line, DC2.4 cells, though vertebrate calf thymus DNA (CT DNA) with less CpG motifs did not. These characteristics differed from mouse peritoneal resident macrophages that do not respond to any naked DNA. The amount of cytokines released from the DC was significantly increased by complex formation with cationic lipids when CpG-motif positive DNAs were used. Unlike murine macrophages or Flt-3 L cultured DC, GM-CSF DC did not release inflammatory cytokines in response to the addition of CT DNA/cationic lipid complex, suggesting that the activation is completely dependent on CpG motifs. Taken together, the results of the present study demonstrate that murine DC produce pro-inflammatory cytokines upon stimulation with CpG-containing DNAs and the responses are enhanced by cationic lipids. These results also suggest that DC are the major cells that respond to naked CpG DNA in vivo, although both DC and macrophages will release inflammatory cytokines after the administration of a DNA/cationic lipid complex.

Animals↗

PubMed2006

Suppression of tumor growth by intratumoral injection of short hairpin RNA-expressing plasmid DNA targeting beta-catenin or hypoxia-inducible factor 1alpha.

To inhibit the growth of murine melanoma B16 cells in mice, we downregulated the gene expression of beta-catenin and hypoxia-inducible factor 1alpha (HIF1alpha) in the tumor cells by delivering short hairpin RNA (shRNA)-expressing plasmid DNA (pDNA) targeting one of these genes. Transfection of any of the shRNA-expressing pDNAs to B16 cells resulted in the reduction of the corresponding mRNA, which was associated with a reduced number of viable cells. A flow cytometric analysis of annexin V labeling assay was also performed to count the number of apoptotic cells. A flow cytometric analysis showed that the suppression of the expression of beta-catenin or HIF1alpha in B16 cells increased the number of apoptotic cells. An intratumoral injection of pshbeta-catenin (shRNA-expressing pDNA targeting beta-catenin) or pshHIF1alpha (shRNA-expressing pDNA targeting HIF1alpha) followed by electroporation greatly suppressed the expression of the corresponding target mRNA in the intradermal tumor tissue. The growth of the intradermal tumor was significantly (P<0.05) suppressed by the treatment. In conclusion, tumor growth was successfully inhibited by the intratumoral delivery of pshbeta-catenin or pshHIF1alpha.

Animals↗

PubMed2006

Fetuin mediates hepatic uptake of negatively charged nanoparticles via scavenger receptor.

We tried to evaluate the possible involvement of fetuin in the scavenger receptors (SRs)-mediated hepatic uptake of polystyrene nanospheres with the size of 50 nm (NS-50), which has surface negative charge (zeta potential=-21.8+/-2.3 mV). The liver perfusion studies in rats revealed that the hepatic uptake of NS-50 pre-coated with fetuin (NS-50-fetuin) was significantly inhibited by poly inosinic acid (poly I), a typical inhibitor of SRs, whereas that of plain NS-50 or NS-50 pre-coated with BSA (NS-50-BSA) was not. The uptake of NS-50-fetuin by cultured Kupffer cells was also significantly inhibited by poly I, and anti-class A scavenger receptors (SR-A) antibody, suggesting that fetuin on NS-50 mediated the recognition and internalization of NS-50 by Kupffer cells and at least SR-A would be responsible for the uptake. Taken that Western blot analysis confirmed that fetuin certainly adsorbed on the surface of NS-50 after the incubation of NS-50 with serum, the results obtained in the present study indicate that fetuin would be one of the serum proteins that were substantially involved in the hepatic uptake of NS-50 via SRs.

Adsorption↗

PubMed2006

Induction of antigen-specific cytotoxic T lymphocytes by immunization with negatively charged soluble antigen through scavenger receptor-mediated delivery.

Antigen-specific cytotoxic T lymphocytes (CTL) are essential for the immunotherapy against cancer or infection diseases although, conventionally, immunization with antigens in soluble form cannot induce CTL. In the present study, we have demonstrated for the first time that ovalbumin (OVA)-specific CTL can be induced without any adjuvants by immunization with soluble OVA with negative charges through scavenger-mediated delivery of antigens to antigen presenting cells (APC). Succinylated, maleylated and aconitylated derivatives were synthesized to allow the introduction of negative charges. All these derivatives can induce OVA-specific CTL and, especially, the CTL activity of mice immunized with maleylated derivatives was comparable with that with OVA emulsified with CFA, known to be the strongest adjuvant. Efficient antigen-specific T cell proliferation and IFN-gamma production were also observed for the OVA derivatives. The OVA derivatives also showed significant protective effects on the growth of OVA-expressing E.G7 tumor cells. In conclusion, the present study demonstrates that the introduction of negative charges to soluble antigens will be a useful strategy for the development of vaccines.

Animals↗

PubMed2006

Incorporation into a biodegradable hyaluronic acid matrix enhances in vivo efficacy of recombinant human interleukin 11 (rhIL11).

In order to improve the therapeutic efficacy of recombinant human interleukin 11 (rhIL11) and to reduce its frequency of administration, the feasibility of a sustained release formulation consisting of hyaluronic acid (HyA) was investigated. rhIL11 was mixed together with an aqueous solution of HyA or HyA and protamine, and the mixture was lyophilized. The resulting powder was compressed into pellet and was subcutaneously administered in the rats. The plasma profile of rhIL11 was determined by ELISA. The mean residence time and t(max) of rhIL11 were much prolonged by administration in an HyA pellet. The additional incorporation of protamine into the formulation further enhanced the plasma duration of the protein. Separately, peripheral platelet counts were measured for several rhIL11-containing solution and pellets. Platelet counts much increased after administration of rhIL11-containing pellets, whereas the effect of bolus subcutaneous administration of rhIL11 solution was limited. The degree of platelet increase in rats treated with the pellets was comparable to that for rats treated with 1- or 2-day continuous infusion from an osmotic mini-pump; these data reflect the importance of increased plasma duration of rhIL11. These data indicate that use of hyaluronic acid as a polymer matrix might enhance the therapeutic efficacy of rhIL11.

Animals↗

PubMed2006

Enhanced antigen-specific antibody production following polyplex-based DNA vaccination via the intradermal route in mice.

DNA vaccination is an attractive approach with various advantages over conventional vaccination. The present study was undertaken to examine whether polyplex-based DNA vaccination could be used to modulate immune responses by plasmid DNA (pDNA). Methylated bovine serum albumin (mBSA) was used as a model of a cationic macromolecular carrier of pDNA encoding obalbumin (OVA) and the effects of polyplex formation of pDNA with mBSA on the antigen-specific immune responses were examined. Anti-OVA IgG antibody production was significantly increased following intradermal immunization with the polyplex compared with naked pDNA, although the induction of cytotoxic T lymphocyte activity was lowered by polyplex formation. We also demonstrated that the disposition and gene expression of pDNA following intradermal injection could be manipulated by polyplex formation. Intriguingly, we also found that the migration of dendritic cells to the injected site could be induced by polyplex formation probably due to a high level of tumor necrosis factor alpha production from the keratinocytes treated with mBSA/pDNA complexeses. Thus, the present study has demonstrated that the immune responses could be biased towards a Th2-type response by polyplex-based DNA vaccination through manipulation of not only pDNA disposition but also dendritic cell migration.

Animals↗

PubMed2006

Moment analysis for kinetics of gene silencing by RNA interference.

RNA interference (RNAi) was quantitatively evaluated from a kinetic viewpoint. A simple kinetic evaluation based on moment analysis was proposed, assuming suppression and recovery phases of gene expression. We defined the area under the curve of the inhibitory effect (AUC(IE)) as an index of the total intensity of RNAi and the mean response time of the inhibitory effect (MRT(IE)) as an index of its duration. The proposed kinetic analysis helps to understand the RNAi effect in a quantitative and time-dependent manner, which will be beneficial for designing RNAi-based gene silencing for both experimental and therapeutic purposes.

Animals↗

PubMed2006

Role of immunostimulatory DNA and TLR9 in gene therapy.

One of the key issues of DNA-based therapies is the immunostimulatory effect caused by DNA, which it has a significant impact on the consequences of these therapies. This is a result of the recognition by Toll-like receptor-9 (TLR9); a pattern recognition receptor for unmethylated CpG motifs within DNA sequences. However, recent studies show that TLR9 can recognize non-CpG motifs and induce cellular activation when DNA is efficiently delivered to TLR9, for example, by cationic lipids. Moreover, recent research shows that TLR9-independent immunoactivation could take place. Here, we have attempted to present an overview of immunoactivation by DNA to optimize DNA-based therapies.

Adenoviridae↗

PubMed2006

Histogram analysis of pharmacokinetic parameters by bootstrap resampling from one-point sampling data in animal experiments.

A bootstrap method is proposed for assessing statistical histograms of pharmacokinetic parameters (AUC, MRT, CL and V(ss)) from one-point sampling data in animal experiments. A computer program, MOMENT(BS), written in Visual Basic on Microsoft Excel, was developed for the bootstrap calculation and the construction of histograms. MOMENT(BS) was applied to one-point sampling data of the blood concentration of three physiologically active proteins ((111)In labeled Hsp70, Suc(20)-BSA and Suc(40)-BSA) administered in different doses to mice. The histograms of AUC, MRT, CL and V(ss) were close to a normal (Gaussian) distribution with the bootstrap resampling number (200), or more, considering the skewness and kurtosis of the histograms. A good agreement of means and SD was obtained between the bootstrap and Bailer's approaches. The hypothesis test based on the normal distribution clearly demonstrated that the disposition of (111)In-Hsp70 and Suc(20)-BSA was almost independent of dose, whereas that of (111)In-Suc(40)-BSA was definitely dose-dependent. In conclusion, the bootstrap method was found to be an efficient method for assessing the histogram of pharmacokinetic parameters of blood or tissue disposition data by one-point sampling.

Algorithms↗

PubMed2005

Inhibition of metastatic tumor growth by targeted delivery of antioxidant enzymes.

To develop effective anti-metastatic therapy, targeted or sustained delivery of catalase was examined in mice. We found that mouse lung with metastatic colonies of adenocarcinoma colon26 cells exhibited reduced catalase activity. The interaction of the tumor cells with macrophages or hepatocytes generated detectable amounts of ROS, and increased the activity of matrix metalloproteinases. Hepatocyte-targeted delivery of catalase was successfully achieved by galactosylation, which was highly effective in inhibiting the hepatic metastasis of colon26 cells. PEGylation, which increased the retention of catalase in the circulation, effectively inhibited the pulmonary metastasis of the cells. To examine which processes in tumor metastasis are inhibited by catalase derivatives, the tissue distribution and proliferation of tumor cells in mice was quantitatively analyzed using firefly luciferase-expressing tumor cells. An injection of PEG-catalase just before the inoculation of melanoma B16-BL6/Luc cells significantly reduced the number of the tumor cells in the lung at 24 h. Daily dosing of PEG-catalase greatly inhibited the proliferation of the tumor cells, and increased the survival rate of the tumor-bearing mice. These results indicate that targeted or sustained delivery of catalase to sites where tumor cells metastasize is a promising approach for inhibiting metastatic tumor growth.

Animals↗

PubMed2005

The uptake and degradation of DNA is impaired in macrophages and dendritic cells from NZB/W F(1) mice.

DNA/anti-DNA Ab immune complexes seem to play the critical roles in the development of systemic lupus erythematosus (SLE). However, little is known about the removal of DNA by MPhi and DC. We found that elicited peritoneal MPhis and BM-derived DCs from a lupus-prone strain of New Zealand Black/White F(1) (NZB/W) mice showed impaired DNA uptake and degradation compared with those from control ICR mice. The impairment was mainly observed as the reduced degradation of DNA probably in endosomal compartment and this impaired DNA degradation might, at least in part, result from the reduced DNA uptake in these phagocytic cells. In addition, these impairments was not related to the disease progression since the cells from diseased, 6-month-old NZB/W mice as well as the cells from prediseased, 5-week-old NZB/W mice also exhibited the similar impairment. We also found that the MPhis and DCs of diseased NZB/W mice showed reduced DNA binding at 4 degrees C. However, this reduced DNA binding could be restored to the control level by pretreatment with DNase. Interestingly, this pretreatment had little effect on the DNA uptake in MPhis and DCs of diseased NZB/W mice at 37 degrees C. Hence, the present results imply an impaired function of lupus MPhis and DCs of NZB/W mice to cause retained DNA clearance.

Animals↗

PubMed2005

Cellular uptake and activation characteristics of naked plasmid DNA and its cationic liposome complex in human macrophages.

Plasmid DNA (pDNA) is an important macromolecular therapeutic agent suitable for DNA-based therapies, such as non-viral gene therapy and DNA vaccination. Unmethylated CpG motifs abundant in bacterial DNA, but not in vertebrate DNA, are known to trigger an inflammatory response, which inhibits transgene expression, while modulating immunological consequences following vaccination. We studied cellular uptake and activation characteristics of naked pDNA and its cationic liposome complex in human macrophage-like cells. The present study has demonstrated that naked pDNA was recognized by human macrophage-like cells via specific mechanisms for polyanions. Moreover, it has shown that pDNA complexed with cationic liposomes activates human macrophage-like cells to induce the production of tumor necrosis factor-alpha (TNF-alpha) in a CpG motif-independent manner, while any types of naked DNA could not induce TNF-alpha production from these cells, regardless of the presence of CpG motifs in pDNA or oligonucleotide (ODN). These findings form an important basis for DNA-based therapies including gene therapy and DNA vaccination.

Cations↗

PubMed2005

Sequence-specific suppression of mdr1a/1b expression in mice via RNA interference.

PURPOSE: RNA interference (RNAi) is a powerful tool for silencing gene expression posttranscriptionally. The purpose of this study was to examine whether in vivo RNAi can be induced against endogenous mdr1a/1b in adult mice and to assess the feasibility of generating P-glycoprotein (P-gp) knockdown mice based on RNAi by a very simple intravenous injection of synthetic small interfering RNA (siRNAs) or siRNA-expressing plasmid DNAs. METHODS: The targeted sequences for silencing mdr1a specifically or mdr1a/1b simultaneously were examined in an in vitro study using a mouse colon carcinoma cell line, colon26 cells, in culture. Mice were repeatedly treated with intravenous synthetic siRNAs or siRNA-expressing plasmid DNAs in naked form administered via a large-volume and high-speed injection, i.e., the hydrodynamics-based procedure. The amount of targeted mRNA and P-gp in the liver were determined by real-time polymerase chain reaction and Western blot analysis, respectively. RESULTS: Among several targeted sequences, two and one optimized sequences were selected for mdr1a and mdr1a/1b, respectively, in the in vitro study. Following administration of synthetic siRNAs or siRNA-expressing plasmid DNAs directed against mdr1a, the mRNA level in the liver was significantly reduced to approximately 50-60% of that in control mice. Furthermore, a slight reduction was observed at the protein level. Similar results were obtained in the experiments using siRNA-expressing pDNA directed against mdr1a/1b. CONCLUSIONS: Our results demonstrate that sequence-specific suppression of mdr1 gene expression is possible at the mRNA level as well as the protein level in mice following intravenous delivery of siRNA effectors.

ATP Binding Cassette Transporter, Subfamily B↗

PubMed2005

Gene silencing in primary and metastatic tumors by small interfering RNA delivery in mice: quantitative analysis using melanoma cells expressing firefly and sea pansy luciferases.

Silencing of oncogenes or other genes contributing to tumor malignancy or progression by RNA interference (RNAi) offers a promising approach to treating tumor patients. To achieve RNAi-based tumor therapy, a small interfering RNA (siRNA) or siRNA-expressing vector needs to be delivered to tumor cells, but little information about its in vivo delivery has been reported. In this study, we examined whether the expression of the target gene in tumor cells can be suppressed by the delivery of RNAi effectors to primary and metastatic tumor cells. To quantitatively evaluate the RNAi effects in tumor cells, mouse melanoma B16-BL6 cells were stably transfected with both firefly (a model target gene) and sea pansy (an internal standard gene) luciferase genes to obtain B16-BL6/dual Luc cells. The target gene expression in subcutaneous primary tumors of B16-BL6/dual Luc cells was significantly suppressed by direct injection of the RNAi effectors followed by electroporation. The expression in metastatic hepatic tumors was also significantly reduced by an intravenous injection of either RNAi effector by the hydrodynamics-based procedure. These results indicate that the both RNAi effectors have a potential to silence target gene in tumor cells in vivo when successfully delivered to tumor cells.

Animals↗

PubMed2005

Cholesterol is required for the polarized secretion of erythropoietin in Madin-Darby canine kidney cells.

It has already been reported that stably expressed exogenous human wild-type EPO (wtEPO) is preferentially secreted to the apical side and one of the three N-linked carbohydrate chains critically acts as an apical sorting determinant in Madin-Darby canine kidney (MDCK) cells. It has been suggested that lipid rafts are involved in the apical sorting of membrane and secretory proteins. To investigate the involvement of lipid rafts in the apical sorting of wtEPO, we examined the effect of cholesterol depletion with methyl-beta-cyclodextrin on the secretion polarity of EPO and analyzed Triton X-100 insoluble cell extracts by sucrose density gradients centrifugation in MDCK cells. We found that wtEPO was shifted in non-polarized direction by cholesterol depletion. Most of the wtEPO was not detectable in the raft fractions by sucrose density gradients centrifugation analysis. These results indicate that apical secretion of EPO involves a cholesterol-dependent mechanism probably not involving lipid rafts.

Animals↗

PubMed2005

Manipulation of local disposition and gene expression characteristics of plasmid DNA following intramuscular administration by complexation with cationic macromolecule.

To modulate the immune responses of DNA vaccine, it is very important to control the disposition and gene expression of plasmid DNA (pDNA) after local administration. We chose methylated bovine serum albumin (mBSA), a cationic macromolecule, as a carrier of pDNA. We examined the effects of complexation of pDNA with mBSA on the disposition and gene expression in mice after intramuscular administration. The elimination from injection site was retarded and the accumulation to lymph nodes was increased at the positively charged mBSA/pDNA complexes. As the charge ratios of mBSA/pDNA complexes were higher, the levels of gene expression were reduced. Antigen specific immune responses were evaluated using pDNA encoding ovalbumin (OVA), pCMV-OVA, as a model antigen-expressing pDNA. However, significant levels of production of anti-ovalbumin IgG antibody were obtained in mice immunized with a positively charged complex, mBSA/pCMV-OVA (8:1) (weight ratio). In vitro experiments using DC2.4 cells, a murine dendritic cell line, demonstrated that the levels of gene expression and cytokine release were increased by complexation. These results suggest that the immune responses might be manipulated by complexation presumably due to the altered disposition and gene expression of pDNA.

Animals↗

PubMed2005

Theoretical considerations involving the pharmacokinetics of plasmid DNA.

Success of in vivo gene therapy relies on the development of gene delivery technologies, by which a well-controlled transgene expression is achieved as far as the spatial and temporal profile of the expression is concerned. Because transgene expression only occurs in cells that are transduced with the gene administered, the tissue distribution of genes is an important factor determining the efficacy of in vivo gene transfer. Plasmid DNA is the simplest vector and its administration in naked or complexed form results in significant transgene expression in various organs. The route of administration, the use of cationic vectors and the administration technique greatly affects the tissue distribution of plasmid DNA and the subsequent transgene expression. Therefore, a clear understanding of the tissue distribution of naked and complexed plasmid DNA is a prerequisite for strategies for developing effective in vivo gene transfer methods. Pharmacokinetics translates the tissue distribution properties of plasmid DNA into quantitative parameters, which can be compared with parameters obtained under different conditions, or with physiological parameters such as blood flow rate. Here we discuss the pharmacokinetic evaluation of the tissue distribution characteristics of plasmid DNA, in the free and complexed forms.

Animals↗

PubMed2005

The hydrodynamics-based procedure for controlling the pharmacokinetics of gene medicines at whole body, organ and cellular levels.

Hydrodynamics-based gene delivery, involving a large-volume and high-speed intravenous injection of naked plasmid DNA (pDNA), gives a significantly high level of transgene expression in vivo. This has attracted a lot of attention and has been used very frequently as an efficient, simple and convenient transfection method for laboratory animals. Until recently, however, little information has been published on the pharmacokinetics of the injected DNA molecules and of the detailed mechanisms underlying the efficient gene transfer. We and other groups have very recently demonstrated that the mechanism for the hydrodynamics-based gene transfer would involve, in part, the direct cytosolic delivery of pDNA through the cell membrane due to transiently enhanced permeability. Along with the findings in our series of studies, this article reviews the cumulative reports and other intriguing information on the controlled pharmacokinetics of naked pDNA in the hydrodynamics-based gene delivery. In addition, we describe various applications reported so far, as well as the current attempts and proposals to develop novel gene medicines for future gene therapy using the concept of the hydrodynamics-based procedure. Furthermore, the issues associated with the clinical feasibility of its seemingly invasive nature, which is probably the most common concern about this hydrodynamics-based procedure, are discussed along with its future prospects and challenges.

Animals↗