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We developed the in vitroimmunization method to induce antigen-specific immune responses in human peripheral blood mononuclear cells (PBMCs). However, when we used a peptide as sensitizing antigen, the antigen-specific immune response was found to be weak, and hence, we could not effectively obtain the antigen-specific antibody gene. In the present study, we attempted to improve the in vitroimmunization method by augmenting the immune response to the peptide antigen. We used a multiple antigen peptide for sensitization. In vitroimmunization of the multivalent antigen elicited a strong antigen-specific immune response in the PBMCs, and we succeeded in obtaining antigen-specific antibody genes by the phage-display method. Further, by combining the variable-region genes and constant-region genes of human IgG, we obtained four independent human monoclonal antibodies specific for tumor necrosis factor-α. This might be a good strategy for generating antigen-specific human monoclonal antibodies using a peptide antigen.

An in vitroimmunization 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 vitroimmunization and phage display. Heavy and light chain variable region genes were obtained from PBMC immunized in vitrowith FcεRIα-expressed KU812F cells. Subsequently a combined phage antibody library 6×103in 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 vitroimmunized PBMCs.

Mild oxidative stress is known to induce premature senescence, termed stress-induced premature senescence (SIPS), in normal human diploid cells. We investigated to determine whether mild oxidative stress would trigger SIPS in a human tumor cell line, human lung adenocarcinoma A549. The results showed that sublethal concentrations of H2O2induced SIPS in A549 cells and consequently attenuated, but did not completely eliminate, the tumorigenicity of these cells. We next investigated the reasons for this incomplete impairment of tumorigenicity in A549 cells in SIPS. The results suggested that H2O2-treated A549 cells are composed of a heterogeneous cell population: one is sensitive to H2O2, and the other is resistant or undergoes reversal; the latter reverted to their original tumorigenic form. The molecular mechanisms determining the cellular fate of tumor cells in SIPS should be identified in order to make use of SIPS and oncogene-induced senescence in tumor cells as methods of tumor suppression.

We constructed a novel cancer-specific regulatable adenoviral expression system comprising two vectors: one expressing rtTA, a reverse tetracycline transactivator regulated by the human telomerase reverse transcriptase (hTERT) gene promoter, the other expressing the target gene regulated by the tetracycline response element (TRE). rtTA transactivates target gene expression in the presence of doxycycline. Using these vectors, we constructed an adenoviral expression system, the Tel-On system, which enables highly efficient target gene transduction. This system enabled efficient and regulatable cancer-specific gene expression, and can be used in targeted cancer gene therapy.

Human monoclonal antibodies have great potential for use in the treatment of various diseases. We have established an in vitroimmunization protocol for inducing antigen-specific antibody production from human peripheral blood mononuclear cells (PBMCs). In the in vitroimmunization 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 vitroimmunization 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 vitroimmunization. These results suggest that the in vitroimmunization protocol is applicable in the generation of an anti-peptide antibody against various antigens, including food allergens, foreign antigens, and self-antigens.

We have established an in vitroimmunization 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 vitroimmunization protocol. P. acneswas 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. acneshence appears to act as an adjuvant in the antibody response in in vitroimmunization.

We previously developed an in vitro immunization (IVI) protocol of human peripheral blood mononuclear cells (PBMC) for generating antigen-specific human antibodies. In order to clarify whether IVI protocolinduces antigen-specific B cell responses in PBMC, we analyzed family gene usage and sequence of the variable region gene of immunoglobulin heavy chain (VH gene) of the antibody produced from the in vitro immunized PBMC. Sequence homology analyses of VH gene demonstrated that a larger repertoire of B cells can be sensitized with mite-extract than with cholera toxin B subunit and rice allergen. Further, antigen-specific B cells were efficiently expanded by using CpG oligodeoxynucleotide as adjuvant. These results suggest that appropriate combination of sensitizing antigen and adjuvant is primarily important for expansion of antigen-specific B cells in IVI protocol.

Previous studies have shown that prostaglandin E2(PGE2) release by splenic F4/80+cyclooxygenase (COX)‐2+macrophages (MØ) isolated from mice, treated with mycobacterial components, plays a major role in the regulation of immune responses. However, splenic MØ, isolated from untreated mice and treated in vitro with lipopolysaccharide and interferon‐γ, express COX‐1 and COX‐2 within 1 day but release only minimal amounts of PGE2following elicitation with calcium ionophore A23187. For further characterization of in vivo requirements for development of PGE2‐releasing MØ (PGE2‐MØ), C57Bl/6 [wild‐type (WT)], and interleukin (IL)‐10‐deficient (IL‐10−/−) mice were treated intraperitoneally with heat‐killed Mycobacterium bovisbacillus Calmette‐Guerin (HK‐BCG). One day following injection, COX‐2 was induced in splenic MØ of both mouse strains. However, PGE2biosynthesis by these MØ was not increased. Thus, expression of COX‐2 is not sufficient to induce PGE2production in vivo or in vitro. In sharp contrast, 14 days after HK‐BCG treatment, PGE2release by COX‐2+splenic MØ increased as much as sevenfold, and a greater increase was seen in IL‐10−/−cells than in WT cells. To further determine whether the 14‐day splenic PGE2‐MØ could be derived from bone marrow precursors, we established a chimera in which bone marrow cells were transfused from green fluorescent protein (GFP)‐transgenic donors to WT mice. Donors and recipients were treated with HK‐BCG simultaneously, and marrow transfusion was performed on Days 1 and 2. On Day 14 after BCG treatment, a significant number of spleen cells coexpressed COX‐2 and GFP, indicating that bone marrow‐derived COX‐2+MØ may be responsible for the increased PGE2production.

Pretreatment with L-leucyl-L-leucine methyl ester (LLME) is a prerequisite for peripheral blood mononuclear cells (PBMCs) to produce antigen-specific antibodies when sensitized with an antigen. Little information, however, is available regarding the mechanisms involved in LLME-induced augmentation of antibody production from PBMCs that are antigen sensitized. In the present study, we attempted to identify the genes involved in the suppression of antibody production from PBMCs that was not treated with LLME, but sensitized with an antigen. Using subtractive screening, we obtained 63 independent genes, including 17 EST genes, that are specific for LLME-nontreated PBMC. Among these genes, the expression of heavy chain ferritin (H-ferritin), CC chemokine ligand 18 (CCL18), and matrix metalloproteinase 12 (MMP12) were augmented in LLME-nontreated PBMCs, suggesting that inflammatory factors might be involved in the suppression of antibody production in LLME-nontreated PBMCs.

Abstract: The cell line D29, which was easily and rapidly established by the promoter-activated production and glutamine synthetase hybrid system, secreted recombinant human interleukin-6 (hIL-6) at a productivity rate of 39.5 μg 10−6 cells day−1, one of the highest reported levels worldwide. The productivity rate was about 130-fold higher than that of the cell line A7, which was established without both promoter activation and gene amplification. Although D29 cells had a high copy number and high mRNA level of the hIL-6 gene as well as a high secretion rate of hIL-6, large amounts of intracellular hIL-6 protein accumulated in D29 cells compared to A7 cells. Northern blotting analysis showed no change in the GRP78/BiP expression level in D29 cells. In contrast, an electrophoresis mobility shift assay revealed strong activation of NF-κB in D29 cells. These results suggest that large amounts of hIL-6 translated from large amounts of hIL-6 mRNA cause excess accumulation of intact hIL-6 in the endoplasmic reticulum (ER), and that subsequent negative feedback signals via the ER overload response inhibit hIL-6 protein secretion. To enhance the hIL-6 productivity rate of D29 cells by releasing the negative feedback signals, the effect of pyrrolidinedithiocarbamate, an inhibitor of NF-κB activation, was examined. Suppression of NF-κB activation in D29 cells produced a 25% augmentation of the hIL-6 productivity rate. Therefore, in highly productive cells like D29 cells, the release of negative feedback signals could increase the total amount of recombinant protein secretion.

An in vitroimmunization (IVI) protocol enables antigen specific antibody production from L-Leucyl-L-Leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBL) upon antigen stimulation in the presence of IL-2, IL-4, and muramyl dipeptide. In the course of our studies, we have found that IL-10 added at the antigen sensitization significantly augmented antibody production level from the LLME-treated PBL. In the present study, we tried to demonstrate the role of IL-10 in the augmentation of antibody production in an IVI protocol by clarifying the cytokine expression profiles in CD4+and CD8+T cells. The results showed that IL-10 skewed the Th1/Th2 balance to Th2-type responses by suppressing Th1-type cytokine production and augmenting Th2-type cytokine production in CD4+and CD8+T cells, as well as in CD19+B cells. Furthermore, IL-10 augmented the expression of CD38, an antigen marker of plasma cells, on B cells, which clearly indicates that IL-10 promoted differentiation and maturation of B cells in an IVI protocol. These results indicate that IL-10 plays an important role in setting the cellular milieu to produce antibodies in an IVI protocol.

Abstract: Human leukemia cell lines, KU812 and KU812F, are immature prebasophillic cell lines and have a potential to differentiate into basophils. Hydrocortisone (HC) and sodium nitroprusside (SNP) can enhance the cell surface FcεRI expression of KU812 cells. However, the differentiated phenotypes of KU812 cells were unstable, hindering the application of KU812 cells to construct a practical invitro allergy reaction evaluation system. Here, we attempted to enhance the cell surface expression of FcεRI on hydrocortisone (HC)- or sodium nitroprusside (SNP)-treated KU812 cells by IgE. The cell surface FcεRI expression was observed in about 20, 20 and 26% of 1 μM HC-, 1 nM SNP- and 450 ng ml-1 IgE-treated KU812 cells, respectively. Whereas, the cell surface FcεRI expression was observed in about 54% of KU812 cells treated with both 450 ng ml-1 IgE and 1 μM HC for 8 days, and in about 33% of KU812 cells treated with both 450 ng ml-1 IgE and 1 nM SNP for 4 days. Ninety five% of the IgE/HC- or IgE/SNP-treated KU812 cells expressed CD 13 antigen, a cell surface marker of basophils. An electrophoretic mobility shift assay revealed that AP-1, NF-AT and NF-κB transcription factors were all activated in IgE/HC- and IgE/SNP-treated KU812 cells. Since the differentiated KU812F cells were more sensitive than KU812 cells for histamine release by sensitization with human IgE and anti-IgE antibody, a practical in vitro allergy reaction evaluation system for general use was constructed using IgE/HC-treated KU812F cells. The differentiated KU812F cells sensitized with an allergicpatient's IgE and mite allergen exhibited histamine release. The constructed in vitro allergy reaction evaluation system using differentiated human leukemia KU812F cells will be useful to study allergic reaction and to analyze physiologically functional substances in allergic disease.

Abstract: We have previously demonstrated that the addition of muramyl dipeptide (MDP), interleukin-2 (IL-2) and IL-4 effectively raises antibody production from L-Leucyl-L-leucine methyl ester (LLME)-treated human peripheral blood lymphocytes (PBLs) against specific soluble antigen when immunized in vitro. However, PBLs from individual donors were separate optimal conditions regarding concentrations for IL-2 and IL-4, which in turn required us to optimize each individual PBLs to effectively produce antigen specific human antibody by in vitro immunization. These individual differences in the requirement for IL-2 and IL-4 reflects the differences in individual immune responses against a specific soluble antigen, which can be elicited by in vitro immunization. In the present study, we investigated these individual differences in the requirement for IL-2 and IL-4 to induce antibody productionin vitro in the PBLs of 12 volunteers (9 healthy donors and 3 allergenic patients). IL-2 requirements for antibody production varied dependent upon each donor, while higher amounts of IL-4 inhibited IgM and IgG production in all of the healthy donors. However, some of the characteristic features for PBLs donated from allergenic included lowered IgM production compared to PBLs derived from healthy donors, and very high IgE production in the absence of cytokines and allergen. These results demonstrate that the sensitivity of PBLs against antigen sensitization differs between healthy donors and atopic patients, which suggests that the frequency of antigen sensitization might be reflected in differing activation states and/or differing subpopulations of lymphocytes in vivo.

Abstract: We have demonstrated that an immature prebasophilic cell line,KU812 cells can be induced to differentiate into basophil-like cells when cultured with hydrocortisone (HC) with enhanced cell surface expression of FcεRI, a high affinity IgE receptor. In this study, we report that sodium nitroprusside (SNP), an intracellular NO donor, also induces cell surface expression of FcεRI on KU812 cells. Cell surface FcεRI expression was detected in about 20% of KU812 cells treated with SNP for 14 days as well as the cells treated with HC for 7 days, while non-treated KU812 cells did not express FcεRI on their cell surface. However, Wright-Giemsa staining and flowcytometry analysis of CD13 and CD15 antigens on HC and SNP treated KU812 cells demonstrated that SNP induced eosinophilic differentiation in KU812 cells differently from HC which induced basophilic differentiation. To further confirm this result, we performed RT-PCR against mRNAs specific for eosinophils, such as eosinophil-derived neurotoxin (EDN) and eosinophil peroxidase(EPO). SNP treated KU812 cells but not HC treated cells expressed EDN and EPO mRNA depending upon the induction of differentiation,clearly demonstrating that SNP induces eosinophilic differentiation in KU812 cells. To clarify that different signaling cascades were activated in HC and SNP treated KU812 cells, we analyzed activities of AP-1, NF-AT and NF-κB transcription factors by EMSA, which are known to be involved in signal transduction pathways downstream from the FcεRI molecule of basophils. All these three transcription factors were activated in HC treated KU812 cells,but not in non-treated and SNP treated KU812 cells. These results indicate that KU812 cells are multi-potent precursor cells which can be induced to differentiate into basophils and eosinophils upon exogenous signals, and that NO is an important factor to decide the eosinophilic differentiation in KU812 cells with enhanced surface expression of FcεRI, and further suggest that different signaling cascades can be activated between basophilic and eosinophilic differentiation in KU812 cells.

In this study, we attempted to establish a novel method of screening anti-allergic lactic acid bacteria (LAB). We cloned the human histidine decarboxylase (HDC) promoter into the promoter-less pPhi-Yellow-RPL-dest1 vector and established KU812F cells transduced with this vector (pHDCp-Phi-Yellow/KU812F). After adding LAB to these cells, the change in fluorescence intensity was monitored by flow cytometry. After screening, we identified several LAB strains that downregulated HDC promoter activity. Functional analysis of these LAB strains indicated that two LAB strains inhibited histamine release from KU812F cells, indicating that this assay system can be used to screen for anti-allergic LAB in a high-throughput manner.

A system is developed to magnetically measure biological antigen-antibody reactions with a superconducting quantum interference device (SQUID) magnetometer. In this system, antibodies are labeled with magnetic nanoparticles of ?-Fe2O3, and the antigen-antibody reactions are measured by detecting the magnetic field from the magnetic nanoparticles. A setup of the system is described, and the sensitivity of the system is studied in terms of detectable weight of nanoparticles. Magnetic particles as small as 600 pg can be detected at present. An experiment is also conducted to measure antigen-antibody reaction with the present system. It is shown that the sensitivity of the present system is better than that of the conventional optical method. A one order of magnitude improvement of sensitivity will be realized by the sophistication of the present system.