Biomedical subjects

Makiya Nishikawa

Publications and source records attributed to Makiya Nishikawa.

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

Analysis of in vivo nuclear factor-kappaB activation during liver inflammation in mice: prevention by catalase delivery.

Nuclear factor-kappaB (NF-kappaB) is a transcription factor that plays crucial roles in inflammation, immunity, cell proliferation, and apoptosis. Until now, there have been few studies of NF-kappaB activation in whole animals because of experimental difficulties. Here, we show that mice receiving a simple injection of plasmid vectors can be used to examine NF-kappaB activation in the liver. Two plasmid vectors, pNF-kappaB-Luc (firefly luciferase gene) and pRL-SV40 (Renilla reniformis luciferase gene), were injected into the tail vein of mice by the hydrodynamics-based procedure, an established method of gene transfer to mouse liver. Then, the ratio of the firefly and R. reniformis luciferase activities (F/R) was used as an indicator of the NF-kappaB activity in the liver. Injection of thioacetamide or lipopolysaccharide plus d-galactosamine increased the F/R ratio in the liver, and this was significantly (P<0.001) inhibited by an intravenous injection of catalase derivatives targeting liver nonparenchymal cells. Imaging the firefly luciferase expression in live mice clearly demonstrated that the catalase derivatives efficiently prevented the NF-kappaB-mediated expression of the firefly luciferase gene. Plasma transaminases and the survival rate of mice supported the findings obtained by the luminescence-based analyses. Thus, this method, which requires no genetic recombination techniques, is highly sensitive to the activation of NF-kappaB and allows us to continuously examine the activation in live animals. In conclusion, this novel, simple, and sensitive method can be used not only for analyzing the NF-kappaB activation in the organ under different inflammatory conditions but also for screening drug candidates for the prevention of liver inflammation.

Animals↗

PubMed2006

Inhibition of adhesion and proliferation of peritoneally disseminated tumor cells by pegylated catalase.

Hydrogen peroxide may aggravate the peritoneal dissemination of tumor cells by activating the expression of a variety of genes. In this study, we used pegylated catalase (PEG-catalase) to examine whether prolonged retention of catalase activity within the peritoneal cavity is effective in inhibiting peritoneal dissemination in mouse models. Murine B16-BL6 cells or colon 26 cells labeled with firefly luciferase gene were inoculated intraperitoneally into syngeneic mice. Compared with unmodified catalase, PEG-catalase was retained in the peritoneal cavity for a long period after intraperitoneal injection. A single injection of PEG-catalase just before tumor inoculation significantly reduced the number of the tumor cells at 1 and 7 days. The changes in the expression of molecules involved in the metastasis were evaluated by real time quantitative PCR analysis. Inoculation of the tumor cells increased the expression of intercellular adhesion molecule (ICAM)-1 in the greater omentum, which was inhibited by PEG-catalase. An injection of PEG-catalase at 3 days after tumor inoculation also reduced the number of the tumor cells, suggesting that processes other than the adhesion of tumor cells to peritoneal organs are also inhibited. Daily doses of PEG-catalase significantly prolonged the survival time of tumor-bearing mice. These results indicate that intraperitoneal injection of PEG-catalase inhibits the multiple processes of peritoneal dissemination of tumor cells by scavenging hydrogen peroxide in the peritoneal cavity.

Abdominal Neoplasms↗

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

Inhibition of peritoneal dissemination of tumor cells by single dosing of phosphodiester CpG oligonucleotide/cationic liposome complex.

Although unmethylated CpG dinucleotide-containing oligodeoxynucleotides (CpG ODN) are able to inhibit tumor metastasis through the induction of antitumor immunity, their stability and delivery to antigen presenting cells needs to be improved. In this study, we formulated a CpG ODN complex with cationic liposomes (CpG ODN-lipoplex) and its antitumor activity was evaluated in peritoneal dissemination models of tumor cells stably labeled with firefly luciferase gene. A single intraperitoneal administration of CpG ODN-lipoplex greatly reduced the number of tumor cells to 0.01% or lower compared with that detected in untreated mice, which may be associated with increased production of TNF-alpha and IL-12. CpG ODN-lipoplex increased the survival time of the tumor-bearing mice, and most long-term survivors rejected rechallenged tumor cells. These results indicate that a single dosing of CpG ODN-lipoplex is effective in inhibiting peritoneal dissemination and inducing long-lasting antitumor immunity.

Abdominal Cavity↗

PubMed2006

PEGylated catalase prevents metastatic tumor growth aggravated by tumor removal.

Although surgical removal is a primary option for treating tumors, it can lead to the increased growth of metastatic tumors. Because surgical procedures may generate reactive oxygen species (ROS), known promoters of tumor metastasis and growth, we investigated whether PEGylated catalase (PEG-catalase, plasma half-life of 13.6 h) was able to prevent this after surgical removal of a footpad tumor in mice. Murine melanoma cells labeled with the firefly luciferase gene were used to monitor the distribution of tumor cells. After inoculation into the footpad, tumor cells were found in the lung, and the number increased with time. The surgical removal of the footpad tumor significantly (p < 0.05) increased the number of metastatic tumor cells and the level of plasma lipoperoxides. An intravenous injection of PEG-catalase significantly (p < 0.05) suppressed the metastatic tumor growth as well as the peroxidation. Quantitative RT-PCR and Western blot analyses indicated that PEG-catalase markedly reduced the increase in the expression of epidermal growth factor receptor. These findings indicate that the removal of tumor produces ROS, which then aggravate metastatic tumor growth by activating several growth factors. PEG-catalase can effectively prevent this metastatic tumor growth by detoxifying the ROS.

Animals↗

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

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

Analysis of the molecular interaction between mannosylated proteins and serum mannan-binding lectins.

The kinetics and specificity of the molecular interaction between proteins modified with varying numbers of mannose residues and isolated rabbit mannan-binding lectin (MBL) were characterized by using surface plasmon resonance spectroscopy (SPR). Mannosylated bovine serum albumin (Man-BSA) with different numbers of mannoses and other mannosylated derivatives of lysozyme (LZM), soybean trypsin inhibitor (STI), superoxide dismutase (SOD) and bovine gamma-immunoglobulin (IgG) were synthesized. Rabbit MBL was isolated by affinity column chromatography and immobilized on the SPR sensor chip via avidin-biotin binding. Binding of Man-BSAs to immobilized rabbit MBL increased with an increase in the number of mannose residues, primarily due to the reduction in dissociation rate. On the other hand, the association rate constant was similar for five mannosylated proteins investigated, whereas the dissociation rate constant differed markedly in spite of the same degree of mannosylation. Specific binding of mannosylated proteins to MBL may depend on the number of mannose residues and their steric configurations.

Animals↗

PubMed2006

Analysis of the molecular interaction of glycosylated proteins with rabbit liver asialoglycoprotein receptors using surface plasmon resonance spectroscopy.

A sensitive, accurate, and efficient biosensor analysis using surface plasmon resonance (SPR) spectroscopy was used for delineating the molecular interaction between rabbit liver asialoglycoprotein receptors (ASGPR) and glycosylated proteins. Isolated rabbit liver ASGPR obtained by affinity column chromatography was dissolved in buffer solution containing TritonX-100 and immobilized on the SPR sensor chip by amine coupling. The SPR study demonstrated that the association rate constants (ka) of galactosylated proteins with ASGPR are dependent on the number of galactose residues, while the dissociation rate constants (kd) are influenced not only by the surface density of the galactose moieties but also by their steric configuration. In addition, it was demonstrated that D-fucosylated BSA had a higher binding affinity to ASGPR than Gal-BSA, when the degree of sugar modification was equivalent.

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

Use of lipoplex-induced nuclear factor-kappaB activation to enhance transgene expression by lipoplex in mouse lung.

BACKGROUND: Although lipofection-induced TNF-alpha can activate nuclear factor kappaB (NF-kappaB), which, in turn, increases the transgene expression from plasmid DNA in which any NF-kappaB responsive element is incorporated, no attempts have been made to use such biological responses as NF-kappaB activation against a vector to enhance vector-mediated gene transfer. METHODS: A lipoplex composed of N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium and cholesterol liposome and plasmid DNA encoding firefly luciferase under the control of the cytomegalovirus immediate early promoter (pCMV-Luc) was intravenously injected into mice. Luciferase activity as well as NF-kappaB activation in the lung were evaluated. Then, a novel plasmid DNA, pCMV-kappaB-Luc, was constructed by inserting 5 repeats of NF-kappaB-binding sequences into the pCMV-Luc. RESULTS: NF-kappaB in the lung was activated by injection of the lipoplex and its nuclear localization was observed. An injection of lipopolysaccharide 30 min prior to the lipofection further activated NF-kappaB. At the same time, the treatment significantly increased the transgene expression by lipoplex, suggesting a positive correlation between expression and NF-kappaB activity. Based on these findings, we tried to enhance the lipoplex-based transgene expression by using NF-kappaB activation. The lipoplex consisting of pCMV-kappaB-Luc showed a 4.7-fold increase in transgene expression in the lung compared with that with pCMV-Luc. CONCLUSIONS: We demonstrated that NF-kappaB activation by lipoplex can be used to enhance lipoplex-mediated transgene expression by inserting NF-kappaB-binding sequences into plasmid DNA. These findings offer a novel method for designing a vector for gene transfer in conjunction with biological responses to it.

Animals↗

PubMed2006

Role of tyrosine and tryptophan in chemically modified serum albumin on its tissue distribution.

To investigate the effect of functional groups in bovine serum albumin (BSA) on its tissue distribution characteristics, tyrosine (Tyr) or tryptophan (Trp) residues of BSA were chemically modified by tetranitromethane (TNM) and 2-hydroxy-5-nitrobenzyl bromide (HNB), respectively. BSA was successfully modified with each reagent depending on the amount of the reagent added to the reaction mixture, and TNM- and HNB-modified BSA derivatives with different degrees of modification were obtained. Circular dichroism measurements showed that slight secondary and large tertiary changes were detectable as the degree of modification increased. After intravenous injection into mice, all synthetic BSA derivatives were eliminated very slowly from the systemic circulation. However, (111)In-TNM(6.6)- and (111)In-HNB(2.0)-BSA, derivatives with a high degree of modification, showed a slightly faster disappearance from the systemic circulation and slightly higher accumulation in the liver than (111)In-unmodified BSA. Pharmacokinetic analyses also demonstrated that the modification of Tyr or Trp residues on BSA had only marginal effects on tissue distribution. These results indicate that the Tyr and Trp residues have little effect on the tissue distribution characteristics of serum albumins, and that the specific modification of these residues may be a promising approach to designing sustained drug delivery systems using serum albumins.

2-Hydroxy-5-nitrobenzyl Bromide↗

PubMed2006

Influence of cholesterol composition on the association of serum mannan-binding proteins with mannosylated liposomes.

In our previous studies, serum mannan-binding protein (MBP) accelerated the uptake by cultured macrophages. The present study was initiated to investigate the kinetics of molecular interaction between mannosylated liposomes and MBP in more details and the effects of lipid composition on the interaction. The analysis was carried out by surface plasmon spectroscopy (SPR) methods, using rabbit serum MBP isolated by affinity chromatography. In SPR studies, neither conventional liposomes nor galactosylated liposomes indicated any interaction, but each mannosylated liposomes had a high response signal corresponding to molecular interaction with immobilized MBP. Association of mannosylated liposomes to serum MBP was not dependent on the lipid composition, suggesting a diffusion-controlled association. Dissociation of the mannosylated liposomes from serum MBP was extremely slow. DSPC/Chol/Man-C4-Chol (90 : 5 : 5, molar ratio) exhibited a slower dissociation rate than DSPC/Chol/Man-C4-Chol (60 : 35 : 5). Clustering of mannose residues on liposomal surfaces might be important in determining the binding affinity of mannosylated liposomes with MBP.

Animals↗

PubMed2006

[Optimization of in-vivo gene transfer through regulating biological response to vectors].

The spatiotemporal distribution of transgenes determines the therapeutic efficacy of in vivo gene transfer. The important parameters of gene transfer are the level, duration, and cell specificity of expression, and the number of transfected cells. Interaction of vectors with blood cells, antigen-presenting cells, serum proteins, and other biological components affects the tissue distribution of vectors and the profile of transgene expression. Although plasmid DNA is less immunogenic than viral vectors, it can induce inflammatory cytokine release, due mainly to the presence of unmethylated CpG dinucleotides (CpG motifs). It was clearly demonstrated that intravenous injection of a plasmid DNA/cationic liposome complex resulted not only in the induction of inflammatory cytokines, but also in the activation of nuclear factor kappaB (NF-kappaB) in the lung. Insertion of additional NF-kappaB-binding sequences into conventional plasmid DNA resulted in a high transgene expression in the lung, suggesting that the biological response to vectors can be used to increase transgene expression. In a marked contrast to this strategy, long-term transgene expression was achieved by reducing the number of the CpG motifs in plasmid DNA. A plasmid encoding murine interferon (IFN)-beta or IFN-gamma with reduced numbers of CpG motifs was highly effective in inhibiting metastatic tumor growth in mice. These results clearly demonstrate the importance of the regulation of biological responses to plasmid vectors to optimize plasmid-based in vivo gene transfer.

Animals↗

PubMed2006

Inhibition of tumour metastasis by targeted delivery of antioxidant enzymes.

Metastasis is one of the most harmful aspects of malignant neoplasm. Interaction of tumour cells with normal cells such as tissue macrophages may generate reactive oxygen species, which would affect various aspects of tumour metastasis. Reactive oxygen species cause damage to both tumour and normal cells and some of them, especially hydrogen peroxide, can also act as intracellular second messengers at sublethal concentrations to increase the transcription of various genes, which can then accelerate the proliferation of tumour cells in metastatic colonies. Therefore, eliminating hydrogen peroxide is one approach to inhibiting tumour metastasis. In this article, the roles of reactive oxygen species in tumour metastasis are reviewed, and the strategies to inhibit tumour metastasis by the targeted delivery of catalase, an enzyme that detoxifies hydrogen peroxide, are discussed.

Animals↗

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

Liver targeting of catalase by cationization for prevention of acute liver failure in mice.

To achieve hepatic delivery of CAT for the prevention of CCl4-induced acute liver failure in mice, two types of cationized CAT derivatives, HMD- and ED-conjugated CAT, were developed. Slight structural changes occurred during cationization and the number of increased free amino groups was 3.1 in HMD-CAT and 13.6 in ED-CAT. 111In-cationized CAT derivatives showed an increased binding to HepG2 cells, and were rapidly taken up by the liver. H2O2-induced cytotoxicity in HepG2 cells was significantly prevented by preincubation of the cells with cationized CAT derivatives. A bolus intravenous injection of the cationized CAT derivatives reduced the hepatotoxicity induced by CCl4 in mice. The ED-CAT, which showed more rapid and greater binding to the liver than the HMD-CAT, exhibited more beneficial effects as far as all the parameters examined (serum GOT, GPT, LDH and hepatic GSH) were concerned, suggesting that a high degree of cationization is effective in delivering CAT to the liver to prevent CCl4-induced hepatotoxicity. These results suggest that cationized CAT derivatives are effective in preventing acute liver failure, and ED-based cationization is a suitable method for developing liver-targetable cationized CAT derivatives, because it provides CAT with a high degree of cationization and a high remaining enzymatic activity.

Animals↗