K. Teruya

Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir

An aqueous extract of Kefir, fermented milk originally produced in the Caucasus mountains, suppressed morphological changes of human melanoma HMV-1 and SK-MEL cells and human normal fibroblastTIG-1 cells caused by UVC-irradiation, suggesting that UV damage can be suppressed by the Kefir extract. The addition of the Kefir extract after UVC-irradiation of HVM-1 cells resulted in a remarkable decrease in intracellular reactive oxygen species (ROS) which had been increased by UVC irradiation. The Kefir extract also stimulated unscheduled DNA synthesis and suppressed UVC-induced apoptosis of HMV-1 cells. A colony formation assay revealed that the Kefir extract rescued HMV-1 cells from cell death caused by UVC irradiation. The Kefir extract, as well as methyl methanethiosulfonate which is known to enhance the nucleotide excision repair (NER) activity, exhibited strong thymine dimer repair-enhancing activity. Epigalocatechin exhibited a weak NER activity but vitamins A, C, and E and catechin showed no NER activity. The thymine dimer repair-enhancing factors in the Kefir extract were heat-stable and assumed to be molecules with a molecular weight of less than 5000. The treatment of HMV-1 cells with the Kefir extract during or before UVC- irradiation also prevented the generation of ROS and thymine dimmer, and suppressed the apoptosis of HMV-1 cells, suggesting that application of Kefir can prevent UV damage.

Erratum to: Suppression of UVC-induced cell damage and enhancement of DNA repair by the fermented milk, Kefir

Under the heading ‘‘Suppression of UVC damage in HMV-1 cells by the addition of the Kefir extract during UVC irradiation,’’ the sentence ‘‘Thymine dimer formation in HMV-1 cells treated with the Kefir extract during UVC-irradiation was considerably reduced 5 h after UVC-irradiation, compared to the control (Fig. 6a).’’ should be replaced by ‘‘Thymine dimer formation in HMV-1 cells treated with the Kefir extract during UVC-irradiation was considerably reduced immediately after UVC-irradiation, compared to the control (Fig. 6a).’’ Under the Fig. 6 legend, the sentences ‘‘(a) and (b): HMV-1 cells (2.0 9 10 cells) were UVC-irradiated with 60 J m in the absence or presence of the Kefir extract (9.6 mg ml). Cells were washed to remove the Kefir extract and then cultured for 5 h. Thymine dimers (a) and apoptotic cells (b) were detected using an immunoblot assay.’’ should be replaced by ‘‘(a) and (b): HMV-1 cells (2.0 9 10 cells) were UVC-irradiated with 60 J m in the absence or presence of the Kefir extract (9.6 mg ml). Cells were washed to remove the Kefir extract. Thymine dimers (a) were detected immediately using an immunoblot assay and apoptotic cells (b) were detected using an immunoblot assay after cultivation for 5 h.’’

Optimization of in Vitro Immunization Protocol to Produce Antigen Specific Human Monoclonal Antibody by Demonstrating the Role of IL-10

We have previously reported that in vitro immunization (IVI) protocol enables antigen specific antibody production from Leu-Leu-Ome (LLME)-treated human peripheral blood lymphocytes (PBL) by stimulating with antigen in the presence of IL-2, IL-4 and muramyl dipeptide (MDP). In the course of our studies to optimize the antibody production, we evaluated the cytokine production profiles in the IVI protocol. IL-10 was produced in non-treated PBL, which was thought to be one of reasons for inability of non-treated PBL to produce antibody, and also produced in LLME-treated PBL 1 wk after the antigen sensitization, where antibody production was strongly enhanced. Furthermore, IL-10 added to the LLME-treated PBL before the antigen sensitization, antibody production decreased. However, IL-10 added to the LLME-treated PBL at the antigen sensitization augmented antibody production level. These results strongly suggest that IL-10 might play an important role in the activation and/or inactivation of T cells, B cells and antigen presenting cells at the antigen sensitization, where IL-10 might alter the cellular milieu, resulting in the suppression or activation of antibody production. In the present study, we tried to further evaluate the function of IL-10 in antibody production in the IVI protocol. Results showed that IL-10 added before the antigen sensitization targets CD8 T cells, which might lead to elicit the suppressive function of T cells, while IL-10 added at the antigen sensitization elicits Th2 type responses via suppressing IFN-gamma and augmenting IL-4 production.

Establishment of Efficient Cloning Method for Variable Region Genes of Antigen Specific Human Monoclonal Antibody

We have developed an in vitro immunization protocol of human peripheral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. By using this protocol, B cells producing antigen-specific antibody can be propagated within a week. In the present study, we tried to establish an efficient strategy to clone variable region genes of antigen-specific human monoclonal antibody by applying in vitro immunized PBMC to the phage display method. To evaluate the efficiency of our strategy, variable region genes prepared by PCR from PBMC immunized in vitro with mite-extract (ME) and non-immunized PBMC were applied to the phage display method. After concentrating the ME-specific phage antibody by several rounds of panning, the number of phage antibodies specific for ME and antigen specificity both increased by using in vitro immunized PBMC. These results demonstrate that efficiency in acquisition of variable region genes of antigen specific human monoclonal antibody can be improved by in vitro immunizing PBMC used as a source of antibody genes.

Production of Human Anti-Peptide Antibody for Clinical Use by In Vitro Immunization

Human monoclonal antibodies have a large potential to be used for diagnosis and therapy of various diseases, such as cancer, allergy, and so on. However, there exist some problems on sensitizing antigens to the human in order to obtain the antigenspecific human antibodies because of ethical and moral reasons. In vitro immunization method (IVI), in which primary B cells are activated to produce antigen-specific B lymphocytes, was established to solve this problem. In our previous study, we have established an IVI of human peripheral blood lymphocytes (PBL) against soluble proteins such as Cholera toxin B subunit (CTB), KLH or Rice allergic protein (RA) (Ichikawa et al., 1999). Furthermore, using a fragment peptide of CTB or RA, we have demonstrated that peptide antigen can be useful as sensitising antigen on IVI. However, some of proteins were difficult to produce antibodies because of serious effect on PBL on IVI, such as cytotoxicity, autoimmune response, change in the cytokine balance, and so on. TNF-α is a pleiotropic cytokine primarily produced by activated macrophages to response to a variety of inflammatory agents, so it is difficult to use as sensitizing antigen on IVI. Furthermore, Anti-TNF-α chimeric monoclonal antibody Infliximab have been reported to improve symptoms of rheumatoid arthritis and Crohns disease, and approved for treatment of these disease (Targan et al., 1997, Maini et al., 1999). In this study, we tried to establish an in vitro immunization protocol against peptide fragments derived from tumor necrosis factor-α (TNF-α) for production of antibody for clinical use.

Production of Anti-Peptide Antibody by In Vitro Immunization

We have established an in vitro immunization method (IVI) of human peripheral blood lymphocytes (PBL) for generating antigen-specific monoclonal antibodies. Until now, we have succeeded in producing monoclonal antibodies specific for a number of soluble antigens. In the present study, we tried to establish an IVI protocol for generating anti-peptide human antibodies. Firstly, we prepared two synthetic peptides from cholera toxin B subunit (pCTB) or rice allergenic protein (pRA). Then we performed IVI of PBL derived from healthy donors by using CTB, RA, and their peptides as the immunized antigens. We measured antibody production levels by a sandwich ELISA. Antigen specificity of produced antibodies were tested by direct ELISA, Western blot and ELISPOT assay. Then we found that anti-CTB antibodies were detected in IVI by using pCTB as antigen. RA-specific antibodies were also generated in IVI against pRA-KLH. In addition, RA-specific B cells were demonstrated to significantly increase by immunization with pRA-KLH, which was evidenced by an ELISPOT assay. These results indicate that anti-peptide antibody can be produced by the IVI method using KLH coupled peptide as sensitizing antigens.

Monocytes Suppress Immune Responses of Peripheral Blood Lymphocytes: Possible Implication of Immature Dendritic Cells

It is impossible to raise antibody production from peripheral blood lymphocytes (PBMCs) against specific soluble antigen when immunized in vitro. However, using PBMCs pretreated with L-Leucyl-L-leucine methyl ester (LLME) before antigen stimulation, it become possible to raise antibody production effectively. In this study, we tried to identify the point of action of LLME, and further to clarify the regulation mechanisms of immune responses in the in vitro immunization. Firstly, we analyzed the population of non-treated or LLME-treated PBMCs by flowcytometry analysis, indicating that monocytes (Mo) and natural killer (NK) cells disappeared in LLME-treated PBMCs as compared with non-treated PBMCs. In these cells, only Mo could downregulate the antibody production from LLME-treated PBMCs. Mo changed its morphology and expression pattern of cell surface antigen after immunization, indicating that differentiation of Mo were induced after antigen stimulation in in vitro immunization. The expression patterns of cell surface maker antigens on the Mo in immunized PBMCs were similar to immature dendritic cell (iDQ differentiated from peripheral blood monocytes with GM-CSF and IL-4 treatment. Furthermore, both Mo in immunized PBMCs and iDC downregulated the antibody production from LLME-treated PBMCs immunized in vitro. These results indicate that Mo downregulated the antibody production of non-treated PBMCs. Moreover, these result suggeste that Mo in immunized PBMCs belongs to iDC, and Mo and iDC might play an important role in suppression of immune responses in PBMCs.

CD8+ T Cells Negatively Regulate the Antibody Production from the Human PBMC being Immunized in Vitro with Antigen

We have developed an in vitro immunization method of human peripheral blood mononuclear cells (PBMC) for generating human antigen-specific antibodies. We have previously demonstrated that the antibody production from the in vitro immunized PBMC was induced only when PBMCs was pretreated with Leu-LeuOMe (LLME), known to be toxic to monocytes and cytolytic cells. In this study, we tried to clarify the point of action of LLME in the in vitro immunization, and further to obtain the larger amount of human antibody with the increased specificity by simplifying the protocol. Firstly, we analyzed the population of PBMC treated with LLME by flowcytometry to investigate the effect of LLME on PBMC, indicating that CD8+ T cells decreased as compared to non-treated PBMC, while CD4+ T cells increased. Furthermore, we analyzed the cell population of in vitro immunized PBMC treated or not treated with LLME, demonstrating that CD8+ T cells decreased when using LLME-treated PBMC in in vitro immunization than when using non-treated PBMC. We have previously reported that LLME-treated PBMC, but not non-treated PBMC had an ability to produce antigen specific antibodies, suggesting that CD8+ T cells negatively regulate the antibody production from human PBMC being immunized in vitro. Actually, PBMC depleted of CD8+ T cells retained the ability to produce antigen specific antibody in the in vitro immunization similarly to the LLME-treated PBMC, although the non-treated PBMC did not produce the antibody. These results demonstrate that CD8+ T cells is a negative regulator for antibody production in the in vitro immunization of human PBMC.

Characterization of Cancer Specific Human Monoclonal IgM Antibodies Produced by Hybridomas Derived from a Patient with Adult T-Cell Leukemia

Adult T cell leukemia (ATL) is a malignant disease characterized by tumorous proliferation of T cells infected with retrovirus human T cell leukemia virus Type-I (HTLV-I). B cell line of TM-1 established by stimulating B cells derived from PBL of ATL patient with LPS produces human IgM antibody specific for HTLV-1-infected cells and can react to broad range of cancer tissues. In this study, we established the hybridomas by fusing TM-1 and the mouse-human heteromyeloma cell line of RF-S1, then characterized the antibodies secreted from the hybridomas. Among these cell lines, by the immunostaining of formalin-fixed tissue sections of colon adenocarcinoma with these monoclonal antibodies three hybridomas named 2E12, 3E9 and 3E10 secreted cancer specific monoclonal IgM antibodies were obtained. Furthermore, we tested for the reactivity of hybridoma clones to the variable cell lines of Jurkat, MOLT-4, Raji, Daudi, Namalwa, KU812, U937 and healthy human PBL by the flowcytometry analysis. 3E9 produced monoclonal antibody react to some of these leukemic cell lines strongly.

Anti-Tumor Activity of Noguchi Catalyser 21™, a Mineral Water Containing Natural Leaf Soil

We evaluated anti-tumor activity of Noguchi Catalyser 21− (Catalyser). Effect of Catalyser was examined on the tumor growth derived from ras/SFME cells transplanted into BALB/c mice. By oral free administration of Catalyser, suppression of the tumor growth was observed since 5th week of tumor transplantation and maximally about 25% at 7th week in comparison with tap water. Furthermore, in order to elucidate this effect cytologically, we evaluated the production of anti-tumor cytokines. RT-PCR analysis revealed that the transcription of IL-12 p40 subunit was significantly enhanced in murine macrophage cell line J774-1, RAW264 and BALB/c mouse-derived peritoneal cavity macrophage. This suggests that enhancement of the tumor immune systems via IL-12 production may be enhanced by Catalyser.