Kosuke Tomimatsu

Extension of the Lifespan of Caenorhabditis elegans by the Use of Electrolyzed Reduced Water

Electrolyzed reduced water (ERW) has attracted much attention because of its therapeutic effects. In the present study, a new culture medium, which we designated Water medium, was developed to elucidate the effects of ERW on the lifespan of Caenorhabditis elegans. Wild-type C. elegans had a significantly shorter lifespan in Water medium than in conventional S medium. However, worms cultured in ERW-Water medium exhibited a significantly extended lifespan (from 11% to 41%) compared with worms cultured in ultrapure water-Water medium. There was no difference between the lifespans of worms cultured in ERW-S medium and ultrapure water-S medium. Nematodes cultured in ultrapure water-Water medium showed significantly higher levels of reactive oxygen species than those cultured in ultrapure water-S medium. Moreover, ERW-Water medium significantly reduced the ROS accumulation induced in the worms by paraquat, suggesting that ERW-Water medium extends the longevity of nematodes at least partly by scavenging ROS.

A rapid and efficient strategy to generate antigen-specific human monoclonal antibody by in vitro immunization and the phage display method.

An in vitro immunization (IVI) protocol was developed for inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). After antigen sensitization of PBMCs by IVI, B cells producing antigen-specific antibody can be propagated within a week. Here, we attempted to establish a rapid, efficient strategy to obtain antigen-specific antibody by the phage display method using in vitro immunized PBMCs. Heavy and light chain variable region genes were easily amplified from these PBMCs immunized with mite extract (ME). After generating a combinatorial phage library (1.6 x 10(5) members), 4 antigen-specific clones were selected by 5 panning rounds using biotinylated antigen and streptavidin magnetic beads. Next, we combined variable region genes of these selected clones with human IgG constant region genes and produced human IgG-type antibody. Direct and competitive enzyme-linked immunosorbent assays demonstrated that the mAb 1C11 clone bound specifically to ME. We thus established a rapid, efficient method to obtain antigen-specific human antibody genes and produce human monoclonal IgG antibody using the phage antibody library generated from in vitro immunized PBMCs.

Production of Human Monoclonal Antibodies against FcεRIα by a Method Combining in Vitro Immunization with Phage Display

An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibodies have great potential, and in particular, efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, FcεRIα, using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with FcεRIα-expressed KU812F cells. Subsequently a combined phage antibody library 6×103 in the size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant FcεRIα and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against FcεRIα. These results suggest that one can obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.

Anti-Peptide Antibody Production Elicited by in Vitro Immunization of Human Peripheral Blood Mononuclear Cells

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitro immunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitro immunization protocol, PBMCs are pretreated with L-leucyl-L-leucine methyl ester (LLME) to remove suppressive cells, and are sensitized and cultured with a soluble antigen in the presence of IL-2, IL-4 and muramyl dipeptide for 8 d, and then an antigen-specific antibody is produced. In this study, we examined the novel possibility of an in vitro immunization protocol, specifically, whether LLME-treated PBMCs can be sensitized with a peptide antigen to produce an anti-peptide antibody. The results indicate that antigen-specific immune responses were elicited by a peptide antigen derived from rice allergen, a cholera toxin B subunit, and TNF-α as a sensitizing antigen in in vitro immunization. These results suggest that the in vitro immunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.

A rapid screening and production method using a novel mammalian cell display to isolate human monoclonal antibodies

Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

Propionibacterium Acnes Acts as an Adjuvant in in Vitro Immunization of Human Peripheral Blood Mononuclear Cells

We have established an in vitro immunization protocol whereby human peripheral blood mononuclear cells (PBMCs) are initially treated with L-leucyl-L-leucine methyl ester (LLME) and subsequently sensitized with antigen in the presence of interleukin (IL)-2, IL-4, and adjuvant. This protocol resulted in the production of antigen-specific antibodies. PBMCs are potentiated to react with exogenous antigens upon treatment with LLME. We are using this system to investigate the immunomodulatory activity of additives. In the present study, we aimed to evaluate the immunomodulatory activity of Propionibacterium acnes (P. acnes), which is known to exhibit various immunomodulatory effects in murine models, using this in vitro immunization protocol. P. acnes was found to augment the production of antigen-specific antibodies by PBMC, possibly through increased production of inflammatory cytokines and/or increased T-B cell interaction. P. acnes hence appears to act as an adjuvant in the antibody response in in vitro immunization.

Antigen-specific in vitro immunization: a source for human monoclonal antibodies.

Human monoclonal antibody has great potential for treatment of various diseases utilizing their specificity against antigens. We have shown an in vitro immunization (IVI) protocol inducing antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs) for efficient production of human monoclonal antibodies. By using IVI method antigen specific antibody genes can be efficiently obtained because of increasing production of antigen-specific antibodies from in vitro immunized PBMCs. This IVI protocol will be widely applied for combination with several display methods and enhance the production of human monoclonal antibodies.

Efficient Production of Human Monoclonal Antibodies by Combining In Vitro Immunization and Phage Display Methods

We have developed an in vitro immunization protocol of human periph- eral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. Upon sensitization of PBMC with antigen in vitro according to the pro- tocol, B cells producing antigen-specific antibody can be propagated within a week. In the present study, we tried to establish a strategy to clone variable region genes of antigen specific human monoclonal antibody by applying in vitro immunized PBMC to the phage display method. By using PBMC immunized in vitro as tem- plate, heavy and light chain variable region genes were easily amplified by PCR. After generating the combinatorial phage library (1.6 × 10 5 members), phage anti- body library was subjected to panning using biotinylated antigen and streptavidin magnetic beads to select antigen-specific phage antibody. After 5 rounds of pan- ning, we obtained 4 antigen-specific clones. We combined variable region genes of these selected clones with human IgG constant region genes, and produced as human IgG format antibody. Among these clones, 1C11 showed a highest reactivity for sensitizing antigen. All these results demonstrate that we could obtain antigen- specific human monoclonal antibody from a relatively small phage antibody library by using in vitro immunized PBMC.

Production of Human Monoclonal Antibodies Against FC Epsilon Receptor I Alpha by Combining In Vitro Immunization with Phage Display

An in vitro immunization protocol using human peripheral blood mononuclear cells (PBMC) was developed to generate human antigen-specific antibodies. Monoclonal antibody has a great potential, especially efficient acquirement of monoclonal antibodies against membrane proteins provides advantages. In this study, we tried to generate a human monoclonal antibody against the high affinity IgE receptor, FcɛRIα, using a method combining in vitro immunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitro with FcɛRIα-expressed KU812F cells. Subsequently a combined phage antibody library of 6 × 103 size was generated. Antigen-specific phage antibody clones were selected by panning with recombinant FcɛRIα and recombined to produce human IgG format antibodies using CHO cells. The antibodies exhibited specific binding against FcɛRIα. These results demonstrated that we could obtain membrane protein-specific human monoclonal antibodies from a relatively small phage antibody library using in vitro immunized PBMCs.