Hiroharu Kawahara

Purification and identification of an IgE suppressor from strawberry in an in vitro immunization system

We purified and identified an IgE suppressor from the strawberry ‘Toyonoka’, based on the decrease of IgE production in in vitro immunization (IVI). Gel filtration experiment indicated that fractions in a 15–48 kDa range and <10 kDa have an IgE suppressive activity. Furthermore, the fraction in 15–48 kDa was subjected to chromatofocusing and found to have activities at isoelectric points, pI 6.0, 7.0, and 8.0–9.2. We focused on the active fractions of pI 8.0–9.2 and the purified a large amount of strawberry extracts by cation exchange resins in batch. A purified 39 kDa protein showed homology to plant glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in N-terminal amino acid sequence and had GAPDH enzymatic activity. Nucleotide sequence and deduced amino acid sequence of the obtained cDNA clone of the protein matched with the sequence of Fragaria x ananassa GAPDH in the GenBank with >98% identical nucleotides and >99% identical amino acids, respectively. The purified strawberry GAPDH suppressed total IgE production in IVI in a dose-dependent manner. From these results, we identified GAPDH as IgE suppressor in the strawberry. Our study may be applicable to the development of new methods to relieve allergic conditions using GAPDH and the screening of other functional factors for human health.

Efficient production of recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium.

A fructose-based cell culture is suitable for the process control of protein production because of slow sugar consumption rate and low lactate accumulation. The fructose transporter, GLUT5, mediates its incorporation into cells and is required for the fructose-based culture. In order to produce efficiently recombinant IgG by metabolic control and co-expression with GLUT5 in a fructose-based medium, an IgG and GLUT5 co-expression vector was constructed and transfected into the human myeloma derived cell line, SC-01MFP, which produced stably recombinant proteins. The cell proliferation in the fructose-based medium was improved by the GLUT5 gene transfection. The recombinant IgG production of the cells cultured in the fructose-based medium exhibited about two-fold increase of that in the glucose-based medium. Flow cytometoric analysis indicated that the GLUT5 protein expression level in cell surface was increased in the fructose-based medium. An exogenous but not endogenous GLUT5 transcription activator remarkably raised IgG productivity in the fructose-based medium when compared to that in the glucose-based medium, suggesting that exogenous GLUT5 expression may be involved in it. The GLUT5 co-expression system may be useful for efficient production of recombinant proteins by the fructose-based cell culture.

Retinoic Acid Improves a Hybridoma Culture in a Fructose-Based Medium by Up-Regulation of Fructose Incorporation via Retinoid Nuclear Receptors

Fructose was focused on as an alternative sugar source to glucose in a hybridoma culture medium because it decreases lactate production during cultivation, leading to cell and product stability. But, not all human hybridoma cell lines grew well in a fructose-based serum-free medium. We found that the addition of all-trans-retinoic acid to the fructose-based medium improved the growth and monoclonal antibody production of hybridoma cell lines by up-regulation of fructose incorporation that represented increased expression of the fructose transporter, GLUT5. Selective activation of retinoid nuclear receptor by synthetic ligands showed that both retinoic acid receptors and retinoid X receptors might be related to the improvement of the fructose-based hybridoma culture. This study might be applicable to cell cultures susceptible to lactate and pH changes as well as hybridoma cultures.

Effective induction and acquisition of human monoclonal IgE antibodies reactive with house-dust mite extracts.

IgE plays a critical role in acute hypersensitivity such as anaphylaxis, asthma, and atopic dermatitis. IgE antibody is, therefore, an essential reagent for studying the mechanisms of these diseases. However, it is difficult to obtain IgE antibody in amounts sufficient for research use because IgE-producing lymphocytes are very rare. To overcome this problem, we investigated the requirements for generating IgE-secreting human hybridomas using in vitro immunization of peripheral blood lymphocytes. First, culture conditions were optimized for IgE production by a combination of the immunomodulatory mediators interleukin-2, interleukin-4, interleukin-6, and muramyl dipeptide. Second, the addition of mite antigen to the cultures resulted in an increased production of antigen-specific IgE as well as antigen-specific IgG and IgM. When activated lymphocytes in these cultures were fused with Burkitt lymphoma cells, ICLU-B, antigen-specific IgE-secreting hybridomas were obtained with high efficiency. These results demonstrate that our culture and in vitro immunization system for human peripheral blood lymphocytes is useful for obtaining antigen-specific IgE.

Induction of cell size vesicles from human lymphoma cell lines and their application to drug carriers

Sodium butyrate (NaB) induced the membrane enclosed cell size vesicles from several IgM producing cell lines. We considered the application of the cell-derived vesicles (CDVs) to drug delivery system (DDS) using the lung cancer specific IgM producing AE6 cell line. Microscopic observation showed that the DiI fluorescence labeled AE6 vesicles were incorporated into the lung cancer cell line A549. The anticancer drug, actinomycin D (actD), contained in AE6 and Ramos vesicles decreased the A549 cell viability to 46 and 62% of control without actD, respectively. The cytotoxic effect in AE6 vesicles was superior to that in the Ramos vesicles that have the lung cancer non-specific IgM on their surfaces. However, the result of the Ramos vesicles suggests that the surface molecules other than IgM may interact with the A549 cells. In our method for vesicle production, more specific and abundant antibodies mounted vesicles can be generated by transfection of their genes into cells followed by NaB treatment. These suggest that the CDVs may be useful for the development of a drug carrier for DDS.

Development of Protein High Expression System by Using Fructose Metabolism

We found that the increased expression of fructose transporter, GLUT5 improved cell proliferation and antibody production in human hybridoma and myeloma cell lines cultured in the fructose-based medium. The IgG and GLUT5 expression vectors cotransfected myeloma clones showed a tendency to increase IgG production in the fructose-based medium more than in the glucose-based medium. The both IgG and GLUT5 expression vector transfected myeloma cells increased its tendency, suggesting that IgG expression might be activated with GLUT5 expression in the fructose-based medium when IgG gene was located in the vicinity of GLUT5 gene.

Characterization of Cell-Sized Vesicles Induced from Human Lymphoid Cell Lines

We developed that induced with a sodium butyrate mass production of cell-size vesicles from several human lymphoma cells. Ramos, a human burkitt lymphoma was the best induction rate among several human cell lines. However, all cells were dead after NaB treatment. The trypan blue dye staining showed the leakless of the vesicle. Therefore, it is suggested that the vesicle could be available for a drug carrier. The immunostaining analysis showed the retainment of surface antigen such as IgM, implying the possibility of specific vesicle production by transfection of desired gene into viable cells followed by the NaB treatment. This study is applicable to membrane protein isolation, drug delivery system, and microreactor development.

Establishment and characterization of new human cell lines for recombinant therapeutic protein production

Background Recombinant therapeutic proteins are increasingly requested with advances in tissue engineering using stem cells. Human cell line is an attractive host for the production of such glycoprotein, but there are few reports on human cells for a commercial production [1]. In this study, we established new human lymphoid cell lines from peripheral blood mononuclear cells (PBMCs) by treatment with phorbol 12-myristate 13-acetate (PMA) under a non-GMP condition, and characterized them by gene and protein expression analyses.

Efficient production of recombinant IgG by the GLUT5 co-expression system.

A fructose containing cell culture medium has the advantage of low lactate production and a small pH change, leading to cell and product stability. But, not all cell lines grow well in the medium, and the fructose transporter, GLUT5, is related to it [1]. Thus, we developed an efficient production system of recombinant proteins by metabolic control and co-expression with GLUT5 in a fructose-based medium [2]. In this report, the availability of the GLUT5 co-expression system was indicated in CHO-K1 and the human cell line, SC-01MFP [3]. As a model, an IgG and GLUT5 co-expression vector was constructed and transfected into cells. When the transfected CHO-K1 and SC-01MFP cells were cultured in the fructose-based medium, both IgG productions were increased up to about two-fold of that cultured in the glucose-based medium (Table ​(Table1).1). Our study may be useful for efficient production of recombinant proteins using the fructose-based cell culture. In particular, the production in SC-01MFP cells is valuable for functional analysis of recombinant proteins with a human glycosylation profile. Table 1 Proliferation and IgG production in the fructose-based medium.

Detection of Anti-Allergic Factors in Strawberry Extracts

We examined the anti-allergic action of strawberry extracts in pollinosis by using an in vitro IgE inducing method. It was found that several kinds of the strawberries suppressed total IgE production as much as epicatechin gallate that is well known as an anti-allergic factor. The IgE decrease was correlated to the decrease of SOCS3 specifically expressed in Th2 cells that promote IgE production. In addition, Biacore analysis revealed that there is a factor with the ability to bind with the ceder pollen allergen Cryj1 in the strawberry extracts. These results suggest that the strawberry extracts may suppress the allergic reaction by acting on both Th2 cells and Cryj1 allergen.