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Biomedical subjects

Hiroyuki Honda

Publications and source records attributed to Hiroyuki Honda.

At least 37 records · Page 2Linked to original sources
PubMed2005

Anticancer effect of hyperthermia on prostate cancer mediated by magnetite cationic liposomes and immune-response induction in transplanted syngeneic rats.

BACKGROUND: The hyperthermic effect of magnetic particles was examined in rat prostate cancer in vivo. Magnetic cationic liposomes (MCLs) have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis losses. METHODS: Rat prostate cancer cells (PLS 10; androgen independent) were injected subcutaneously into the flank of F344 rats. MCLs were injected into rat prostate cancer nodules that had grown to 5-6 mm in diameter, and were then exposed to an AMF. Tumor growth rates were measured. To examine whether hyperthermia caused immune induction for PLS 10, cytotoxicity assays and immunohistochemical staining for CD3, CD4, CD8, and Heat Shock Protein (HSP) 70 were performed. RESULT: The tumor temperature increased to 45 degrees C whereas the body temperature remained at around 38 degrees C. Tumor regression was observed in the hyperthermic group. CD3, CD4, and CD8 immunocytes were present in the tumor tissues of the rats exposed to hyperthermia, but they were not detected in any of the tumor tissue of untreated rats. HSP70 also appeared in the viable area at its boundary with the necrotic area. The cytotoxic activity of tumor-transplanted rats for PLS 10 cells increased in hyperthermic-treatment rats. CONCLUSION: These results suggest that hyperthermia using MCLs is an effective therapy for prostate cancer, since this treatment appears to kill the prostate cancer cells not only directly by heating but also by inducing an immune response. This therapy may cure not only the primary lesion but also metastatic lesions.

Animals↗
PubMed2005

Cancer immunotherapy based on intracellular hyperthermia using magnetite nanoparticles: a novel concept of "heat-controlled necrosis" with heat shock protein expression.

Heat shock proteins (HSPs) are highly conserved proteins whose syntheses are induced by a variety of stresses, including heat stress. Since the expression of HSPs, including HSP70, protects cells from heat-induced apoptosis, HSP expression has been considered to be a complicating factor in hyperthermia. On the other hand, recent reports have shown the importance of HSPs, such as HSP70, HSP90 and glucose-regulated protein 96 (gp96), in immune reactions. If HSP expression induced by hyperthermia is involved in tumor immunity, novel cancer immunotherapy based on this novel concept can be developed. In such a strategy, a tumor-specific hyperthermia system, which can heat the local tumor region to the intended temperature without damaging normal tissue, would be highly advantageous. To achieve tumor-specific hyperthermia, we have developed an intracellular hyperthermia system using magnetite nanoparticles. This novel hyperthermia system can induce necrotic cell death via HSP expression, which induces antitumor immunity. In the present article, cancer immunology and immunotherapy based on hyperthermia, and HSP expression are reviewed and discussed.

Animals↗
PubMed2005

Novel strategy for protein exploration: high-throughput screening assisted with fuzzy neural network.

To engineer proteins with desirable characteristics from a naturally occurring protein, high-throughput screening (HTS) combined with directed evolutional approach is the essential technology. However, most HTS techniques are simple positive screenings. The information obtained from the positive candidates is used only as results but rarely as clues for understanding the structural rules, which may explain the protein activity. In here, we have attempted to establish a novel strategy for exploring functional proteins associated with computational analysis. As a model case, we explored lipases with inverted enantioselectivity for a substrate p-nitrophenyl 3-phenylbutyrate from the wild-type lipase of Burkhorderia cepacia KWI-56, which is originally selective for (S)-configuration of the substrate. Data from our previous work on (R)-enantioselective lipase screening were applied to fuzzy neural network (FNN), bioinformatic algorithm, to extract guidelines for screening and engineering processes to be followed. FNN has an advantageous feature of extracting hidden rules that lie between sequences of variants and their enzyme activity to gain high prediction accuracy. Without any prior knowledge, FNN predicted a rule indicating that "size at position L167," among four positions (L17, F119, L167, and L266) in the substrate binding core region, is the most influential factor for obtaining lipase with inverted (R)-enantioselectivity. Based on the guidelines obtained, newly engineered novel variants, which were not found in the actual screening, were experimentally proven to gain high (R)-enantioselectivity by engineering the size at position L167. We also designed and assayed two novel variants, namely FIGV (L17F, F119I, L167G, and L266V) and FFGI (L17F, L167G, and L266I), which were compatible with the guideline obtained from FNN analysis, and confirmed that these designed lipases could acquire high inverted enantioselectivity. The results have shown that with the aid of bioinformatic analysis, high-throughput screening can expand its potential for exploring vast combinatorial sequence spaces of proteins.

Fuzzy Logic↗
PubMed2005

Magnetic force-based mesenchymal stem cell expansion using antibody-conjugated magnetoliposomes.

Recently, there has been an accumulation of evidence indicating that human mesenchymal stem cells (MSCs, multipotent cells resident in the bone marrow) are useful for autologous cell transplantation. However, only small numbers of MSCs have been obtained in bone marrow aspirates. We have developed a novel methodology for enriching and proliferating MSCs from bone marrow aspirates using antibody-conjugated magnetoliposomes (AMLs). The AMLs are liposomes conjugated to anti-CD105 antibody (immunoliposomes) and contain magnetite nanoparticles (diameter 10 nm). In the present study, the AMLs were added to a small volume (1 mL) of human bone marrow aspirate. After a 1-h incubation period, the bone marrow aspirates containing AMLs were seeded into 10-cm tissue culture dishes, and a disk-shaped magnet (diameter 2.2 cm; height 1 cm; 4000 Gauss) was positioned under the dish to enrich MSCs by magnetic force. The MSCs proliferated, forming colonies at the site where the magnet was positioned. In contrast, no colonies and very few viable cells were observed in ordinary culture based on plastic-adherent tendencies of cells without use of AMLs. These results suggest that this AML culture method can rapidly and efficiently expand a small number of MSCs into numbers suitable for clinical application.

Animals↗
PubMed2005

Association between Helicobacter pylori seropositivity and NAD(P)H:quinone oxidoreductase 1 (NQO1) C609T polymorphism observed in outpatients and health checkup examinees.

BACKGROUND: Significant associations of Helicobacter pylori (H. pylori) seropositivity have been found with several host polymorphisms. This study investigated the associations of functional polymorphisms of the NQO1, GSTM1, and GSTT1 genes of detoxification enzymes, with the seropositivity, as well as with pepsinogen levels, as markers of gastric atrophy. METHODS: The subjects were 241 noncancer outpatients who had participated in an H. pylori eradication program (HPE) at Aichi Cancer Center Hospital, and 465 health checkup examinees in Nagoya (HCE). The NQO1 C609T, GSTM1, and GSTT1 polymorphisms were determined by triplex polymerase chain reaction with confronting two-pair primers (PCR-CTPP). RESULTS: The sex- and age-group-adjusted odds ratio (OR) of NQO1 C/C for H. pylori seropositivity relative to T/T was highly significant; OR, 1.92; 95% confidence interval (95% CI), 1.22-3.03. The ORs of the GSTM1 present type and GSTT1 present type for H. pylori seropositivity were not significant; OR, 0.87; 95% CI, 0.64-1.20 and OR, 1.14; 95% CI, 0.83-1.57, respectively. The association of the NQO1 C/C genotype with H. pylori seropositivity was observed only for never-smokers; OR, 2.25; 95% CI, 1.33-3.79. The genotypes of the NQO1, GSTM1, and GSTT1 genes were not associated with the development of atrophic gastritis among the H. pylori-seropositive subjects. CONCLUSION: This is the first study to report a significant association of the NQO1 C609T polymorphism with H. pylori seropositivity. The biological mechanism explaining the significant association with the seropositivity remains to be elucidated.

Adult↗
PubMed2005

The effect of RGD peptide-conjugated magnetite cationic liposomes on cell growth and cell sheet harvesting.

Tissue engineering requires novel technologies for establishing 3D constructs, and the layered method of culturing cell sheets (cell sheet engineering) is one potentially useful approach. In the present study, we investigated whether coating the culture surface with RGD (Arg-Gly-Asp) peptide-conjugated magnetite cationic liposomes (RGD-MCLs) was able to facilitate cell growth, cell sheet construction and cell sheet harvest using magnetic force without enzymatic treatment. To promote cell attachment, an RGD-motif-containing peptide was coupled to the phospholipid of our original magnetite cationic liposomes (MCLs). The RGD-MCLs were added to a commercially available 24-well ultra-low-attachment plate the surface of which comprised a covalently bound hydrogel layer that was hydrophilic and neutrally charged. A magnet was placed on the underside of the well in order to attract the RGD-MCLs to the surface of the well, and then NIH/3T3 cells were seeded into the well. Cells adhered to the bottom of the culture surface, which was coated with RGD-MCLs, and the cells spread and proliferated to confluency. After incubation, the magnet was removed and the cells were detached from the bottom of the plates, forming a contiguous cell sheet. Because the sheets contained magnetite nanoparticles, they could be harvested using a magnet inserted into the well. These results suggest that this novel methodology using RGD-MCLs and magnetic force, which we have termed 'magnetic force-based tissue engineering (Mag-TE)', is a promising approach for tissue engineering.

Adsorption↗
PubMed2005

Discovery of glpC, an organic solvent tolerance-related gene in Escherichia coli, using gene expression profiles from DNA microarrays.

Gene expression profiles were collected from Escherichia coli strains (OST3410, TK33, and TK31) before and after exposure to organic solvents, and the six genes that showed higher gene expression were selected. Among these genes, glpC encoding the anaerobic glycerol-3-phosphate dehydrogenase subunit C remarkably increased the organic solvent tolerance.

Cyclohexanes↗
PubMed2005

The gene yggE functions in restoring physiological defects of Escherichia coli cultivated under oxidative stress conditions.

DNA microarray analysis showed that yfiD, yggB, and yggE genes were up-regulated when superoxide dismutase (SOD)-deficient Escherichia coli IM303 (I4) was cultivated under the oxidative stress generated by photoexcited TiO(2), and pYFD, pYGB, and pYGE were constructed by inserting the respective genes into a pUC 19 vector. The content of reactive oxygen species (ROS) in IM303 (I4) cells carrying pYGE was reduced to 31% of ROS content in the control cells with pUC 19. In the culture of wild-type strain, E. coli MM294, in the medium with paraquat (10 micromol/l), maximum specific growth rate of the cells with pYGE was about five times higher than that of the control cells, with a decreased ROS content in the former cells. The introduction of pYGE also suppressed the occurrence of the cells with altered amino acid requirement in the culture of MM294 cells with paraquat.

Escherichia coli↗
PubMed2005

Medical application of functionalized magnetic nanoparticles.

Since magnetic particles have unique features, the development of a variety of medical applications has been possible. The most unique feature of magnetic particles is their reaction to a magnetic force, and this feature has been utilized in applications such as drug targeting and bioseparation including cell sorting. Recently, magnetic nanoparticles have attracted attention because of their potential as contrast agents for magnetic resonance imaging (MRI) and heating mediators for cancer therapy (hyperthermia). Magnetite cationic liposomes (MCLs), one of the groups of cationic magnetic particles, can be used as carriers to introduce magnetite nanoparticles into target cells since their positively charged surface interacts with the negatively charged cell surface; furthermore, they find applications to hyperthermic treatments. Magnetite nanoparticles conjugated with antibodies (antibody-conjugated magnetoliposomes, AMLs) are also applied to hyperthermia and have enabled tumor-specific contrast enhancement in MRI via systemic administration. Since magnetic nanoparticles are attracted to a high magnetic flux density, it is possible to manipulate cells labeled with magnetic nanoparticles using magnets; this feature has been applied in tissue engineering. Magnetic force and MCLs were used to construct multilayered cell structures and a heterotypic layered 3D coculture system. Thus, the applications of these functionalized magnetic nanoparticles with their unique features will further improve medical techniques.

Animals↗
PubMed2005

Heat immunotherapy using magnetic nanoparticles and dendritic cells for T-lymphoma.

Dendritic cells (DCs) are potent antigen-presenting cells that play important roles in regulating immune responses in cancer. Immunotherapy using these immunocytes has become an accepted therapeutic modality. We previously reported that hyperthermia using magnetic nanoparticles induces antitumor immunity, which could be activated by adjuvant including cytokines. In the present study, we investigated the therapeutic effects of hyperthermia combined with DC immunotherapy on mouse EL4 T-lymphoma. Magnetite cationic liposomes (MCLs) have a positive surface charge and generate heat in an alternating magnetic field (AMF) due to hysteresis loss. MCLs were injected into an EL4 nodule in C57BL/6 mice, which were subjected to AMF for 30 min. The temperature at the surface of the tumor reached 45 degrees C and was maintained by controlling the magnetic field intensity. Hyperthermia treatment was repeated twice with 24 h intervals. After hyperthermia, immature DCs were directly injected into the EL4 nodule. As a result, complete regression of tumors in 75% (6/8) of the mice was observed, while the percentage of complete regression of tumors was 12.5% (1/8) in the case of mice treated by hyperthermia alone. This novel cancer therapy, which we have termed "heat immunotherapy", may be applicable to patients with advanced malignancies.

Animals↗
PubMed2005

Screening of cytokines to enhance vaccine effects of heat shock protein 70-rich tumor cell lysate.

Heat shock proteins (HSPs) have been recognized as significant participants in immune reactions. We have previously reported that heat-treated cells expressing HSP70 can mediate potent antitumor immune responses. As successful immunotherapy is dependent on the host immune system, the present study evaluated whether systemic administration of immunocyte stimulatory and growth promoting cytokines could enhance heat-treated cell lysate vaccine (HCLV) immunization to further promote the antitumor immunity. After heating mouse melanoma B16 cells (43 degrees C, 30 min) to elicit increased HSP70 expression, cells were lysed by freeze thawing to prepare HCLV. In approaches using a poorly immunogenic melanoma B16, the effects of various cytokines (IL-1beta, -2, -4, -6 and -12, IFN-beta and -gamma, GM-CSF and TNF-alpha) were assessed in combination with HCLV. Syngenic C57BL/6 mice were immunized subcutaneously with HCLV twice, on days -14 and -7, while cytokines were injected intraperitoneally on day -7. Subcutaneous B16 cell challenge was performed on day 0. IL-12 significantly enhanced the efficacy of HCLV, compared to non-heated cell lysate vaccine (CLV) and non-vaccination. Systemic administration of recombinant IL-12 augmented the efficacy of HCLV, inducing protective immunity against tumor challenge and enhancing cytotoxicity assessed in primed splenocytes against B16 cells in treated mice. These results suggest that IL-12 represents an important modulator of antitumor immune responses induced by HCLV, and may facilitate further efforts to develop novel cancer immunotherapies based on HSP70-mediated vaccination.

Animals↗
PubMed2005

Three-dimensional high-density culture of HepG2 cells in a 5-ml radial-flow bioreactor for construction of artificial liver.

A three-dimensional high-density cell culture is essential for the construction of an artificial tissue. Many researchers have reported that three-dimensional cell culture enhances cell function. The use of a radial-flow bioreactor (RFB) has enabled the cultivation of cells at high density for constructing a three-dimensional tissue. In this study, we have developed a novel, small RFB, which has a bed volume of 5 ml and is equipped with a porous support as an immobilized scaffold; its performance was tested using the hepatoblastoma cell line, HepG2. Among the other supports tested here, hydroxyl apatite was selected from the viewpoint of its ability to support good cell growth at high density with uniform distribution in a bioreactor. The HepG2 cells grew well in the scaffold under a sufficient supply of nutrients by radial flow and were used to construct a three-dimensional tissue in the scaffold. The concentration of the cells cultivated in this 5-ml RFB reached 10(8) cells/ml and the glucose consumption rate was almost similar to that obtained when using a 30-ml RFB, which has already been reported previously. This high glucose consumption continued over 7 d after the growth phase. Furthermore, albumin production was maintained in the stable phase. Gene expression profiles of cells obtained from long-term cultures in the 5-ml RFB were analyzed. It was found that the expressions of genes encoding the cell cycle-related proteins, cyclins, and cell cycle division 2 (cdc2) were suppressed in the stable phase. In addition, the number of cells incorporating 5'-bromo-2'-deoxyuridine (BrdU) in the stable phase markedly decreased compared with that in the growth phase. These results indicated that the majority of cells in the stable phase remain in the G0/G1 phase. Furthermore, this implies that the three-dimensional tissue constructed in the 5-ml RFB showed the high function similar to a normal liver in the human body. Therefore, the 5-ml RFB was considered as a useful tool and a substitute method for animal experiments.

Bioreactors↗
PubMed2004

Artificial neural network approach for selection of susceptible single nucleotide polymorphisms and construction of prediction model on childhood allergic asthma.

BACKGROUND: Screening of various gene markers such as single nucleotide polymorphism (SNP) and correlation between these markers and development of multifactorial disease have previously been studied. Here, we propose a susceptible marker-selectable artificial neural network (ANN) for predicting development of allergic disease. RESULTS: To predict development of childhood allergic asthma (CAA) and select susceptible SNPs, we used an ANN with a parameter decreasing method (PDM) to analyze 25 SNPs of 17 genes in 344 Japanese people, and select 10 susceptible SNPs of CAA. The accuracy of the ANN model with 10 SNPs was 97.7% for learning data and 74.4% for evaluation data. Important combinations were determined by effective combination value (ECV) defined in the present paper. Effective 2-SNP or 3-SNP combinations were found to be concentrated among the 10 selected SNPs. CONCLUSION: ANN can reliably select SNP combinations that are associated with CAA. Thus, the ANN can be used to characterize development of complex diseases caused by multiple factors. This is the first report of automatic selection of SNPs related to development of multifactorial disease from SNP data of more than 300 patients.

Alleles↗
PubMed2004

Magnetite nanoparticle-loaded anti-HER2 immunoliposomes for combination of antibody therapy with hyperthermia.

Anti-HER2 antibody can induce antitumor responses, and can be used in delivering drugs to HER2-overexpressing cancer. Previously, we produced hyperthermia using magnetite nanoparticles that generate heat in an alternating magnetic field. In the present study, we constructed anti-HER2 immunoliposomes containing magnetite nanoparticles, which act as tumor-targeting vehicles, combining anti-HER2 antibody therapy with hyperthermia. The magnetite nanoparticle-loaded anti-HER2 immunoliposomes exerted HER2-mediated antiproliferative effects on SKBr3 breast cancer cells in vitro. Moreover, 60% of magnetite nanoparticles were incorporated into SKBr3, and the cells were then heated at 42.5 degrees C under an alternating magnetic field, resulting in strong cytotoxic effects. These results suggest that this novel therapeutic tool is applicable to treatment of HER2-overexpressing cancer.

Antibodies, Monoclonal↗
PubMed2004

Construction of robust prognostic predictors by using projective adaptive resonance theory as a gene filtering method.

MOTIVATION: For establishing prognostic predictors of various diseases using DNA microarray analysis technology, it is desired to find selectively significant genes for constructing the prognostic model and it is also necessary to eliminate non-specific genes or genes with error before constructing the model. RESULTS: We applied projective adaptive resonance theory (PART) to gene screening for DNA microarray data. Genes selected by PART were subjected to our FNN-SWEEP modeling method for the construction of a cancer class prediction model. The model performance was evaluated through comparison with a conventional screening signal-to-noise (S2N) method or nearest shrunken centroids (NSC) method. The FNN-SWEEP predictor with PART screening could discriminate classes of acute leukemia in blinded data with 97.1% accuracy and classes of lung cancer with 90.0% accuracy, while the predictor with S2N was only 85.3 and 70.0% or the predictor with NSC was 88.2 and 90.0%, respectively. The results have proven that PART was superior for gene screening. AVAILABILITY: The software is available upon request from the authors. CONTACT: honda@nubio.nagoya-u.ac.jp

Algorithms↗
PubMed2004

Hyperthermia using magnetite cationic liposomes for hamster osteosarcoma.

BACKGROUND: We have developed magnetite cationic liposomes (MCLs) and applied them to local hyperthermia as a mediator. MCLs have a positive charge and generate heat under an alternating magnetic field (AMF) by hysteresis loss. In this study, the effect of hyperthermia using MCLs was examined in an in vivo study of hamster osteosarcoma. METHOD: MCLs were injected into the osteosarcoma and then subjected to an AMF. RESULTS: The tumor was heated at over 42 degrees C, but other normal tissues were not heated as much. Complete regression was observed in 100% of the treated group hamsters, whereas no regression was observed in the control group hamsters. At day 12, the average tumor volume of the treated hamsters was about 1/1000 of that of the control hamsters. In the treated hamsters, no regrowth of osteosarcomas was observed over a period of 3 months after the complete regression. CONCLUSION: These results suggest that this treatment is effective for osteosarcoma.

Journal Article↗
PubMed2004

Angiotensin II inhibitory peptide found in the receptor sequence using peptide array.

Peptide array consisting of hundreds of peptides spatially addressed and synthesized on a cellulose membrane support was used to screen ligand-inhibitory peptides. As a model, angiotensin II (Ang II), a significant peptide related to the treatment of cardiovascular diseases, was chosen as the target ligand. Peptide arrays covering the Ang II receptor type 1 sequence were prepared, and peptide domains with high affinity to the Ang II fluorescein conjugate were investigated. The peptide (VVIVIY) within the first transmembrane region exhibited the highest affinity to Ang II. The synthesized soluble VVIVIY peptide had an 84% inhibitory effect on Ang II-induced aorta contraction. These results indicate that our screening strategy utilizing peptide array is an effective approach for the peptide drug development.

Amino Acid Sequence↗