Human-human Hybridoma HB4C5 Cells Research Articles

Screening of Immunostimulatory Activity from Indonesian Kampung (Gallus domesticus) Egg White Water Extract: in vitro study

The most popular Indonesian native chicken, known as kampung chicken, is maintained under free-range conditions therefore it is prone to high environmental stress. Indonesian native chicken and its product are always regarded as having health benefits compared to commercial chicken by Indonesian society. But there is still limited report regarding Indonesian native chicken and its product. This study focused on screening immunostimulatory activity from Indonesian native chicken egg white using in vitro approaches as a functional food. Indonesian native chicken egg white (NEW) was extracted using distilled water supplemented to human-human hybridoma HB4C5 cells to examine the IgM production stimulating activity using ELISA. The gene expression was also examined using qRT-PCR. The ability of NEW on stimulating immunoglobulin production by mouse splenocytes was analyzed. Commercial egg white water extract (CEW) was used as a comparison. The data were analyzed using One-way ANOVA and continued by using post-hoc analysis using Tukey’s multiple comparison test. The results showed that NEW and CEW modulated IgM production by the HB4C5 cells 8.72-fold and 6.75-fold, respectively, compared to control. NEW stimulated immunoglobulin (Ig) production by the mouse splenocytes higher than CEW. To conclude, NEW provides an immunostimulating activity that can potentially act as a health-promoting food.

Immunostimulatory effect of ikan peda (fermented Rastrelliger sp.) water extract on IgM production by HB4C5 cells

Ikan peda is one of Indonesia’s traditional fermented food products. It uses mackerel fish (Rastrelliger sp.), also known as ikan kembung in Indonesia, as its main ingredient. It is made through fermentation by adding high concentration of salt in order to extend the shelf-life and bring out the desired flavor of the fish. Although fermented foods are known to be beneficial towards health, fewer studies have been reported regarding the actual health promoting potential of Indonesian fermented foods, especially ikan peda. Thus, the immunostimulatory effects of ikan peda extract were examined. Water-extract of ikan peda was tested to human-human hybridoma HB4C5 cells. It was found that ikan peda extract stimulates the production of IgM by HB4C5 cells according to ELISA results. Data from real-time RT-PCR result showed that ikan peda extract has the ability to increase IgM gene expression by 3-fold at the highest concentration treatment. Results from the heat and enzyme treatments showed that the active substance contained in the ikan peda extract was expected to be a heat-stable protein. Although, the active substance and the mechanism is yet to be determined, and can be considered for future experiments. Our findings suggest that consumption of ikan peda would contribute to the promotion of health.

Lactate dehydrogenase enhances immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Lactate dehydrogenase (LDH) derived from rabbit muscle enhanced IgM production by human-human hybridoma HB4C5 cells 12.4-fold at 320 mug/ml under serum-free conditions. LDHs from pig muscle and pig heart also accelerated IgM production 8.4- and 6.4-fold, respectively. The immunoglobulin production stimulating activity of LDH was not accompanied by activation of cell proliferation. LDH from rabbit muscle facilitated IgM and IgG production by human peripheral blood lymphocytes. This means LDH stimulates immunoglobulin production not only by the specified hybridoma cell line, but also by unspecified immunoglobulin producers. LDH from rabbit muscle enhanced IgM production of transcription-suppressed HB4C5 cells treated with actinomycin D. The immunoglobulin production-stimulating factors (IPSFs) effect of LDH was slightly weakened by sodium fluoride (translation inhibitor) treatment of HB4C5. Moreover, the amount of intracellular IgM of monensin-treated HB4C5 cells was obviously enhanced by LDH. This result means that the IPSF effect of LDH is irrelevant to the post-translation activity of target cells. It is expected from these findings that LDH from rabbit muscle accelerates the translation step to enhance immunoglobulin productivity. The immunoglobulin production-stimulating activity of LDH was inhibited by colchicine, endocytosis inhibitor. This fact suggests that it is necessary for LDH to be taken by target cells to act as an IPSF.

High IgM production by human-human hybridoma HB4C5 cells cultured in microtubes.

HB4C5 cells, a human-human hybridoma, were cultured in microtubes. After 24 h of cultivation, growth of the cells cultured in microtubes was 1.2- to 1.5-fold that in culture dishes or 96-well culture plates. The production of IgM was 2.6- to 3.3-fold that in the 96-well culture plates.

Increase of immunoglobulin productivity of human-human hybridoma HB4C5 cells by histone.

A histone mixture (H1, H2A, H2B, H3, and H4) derived from calf thymus stimulated IgM production by human-human hybridoma HB4C5 cells. On the contrary, the histone mixture did not increase IgM production by the human Burkitt's lymphoma cell line NAT-30, IgG production by the human B lymphoblastoid cell line HMy-2, and IgE production by the human myeloma cell line U266. The immunoglobulin production-stimulating activity of the histone mixture was inactivated by trypsin or chymotrypsin digestion. In addition, confocal laser microscopic analysis had shown that HB4C5 cells incorporated a lot of histone but other cell lines did not incorporate it as much. These facts strongly suggest that histone acts as an immunoglobulin production-stimulating factor (IPSF) after internalization into the human B cell lines and the native structure of histone is required for the IPSF activity.

Aldolase from rabbit muscle enhances immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

d-fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) derived from rabbit muscle facilitated immunoglobulin production by human-human hybridoma HB4C5 cells under serum-free condition. IgM production by HB4C5 cells was stimulated by the addition of aldolase from rabbit muscle at the concentrations of more than 100 μg ml −1. The enzyme also enhanced IgM and IgG production by human peripheral blood lymphocytes about 5.3-fold and 1.4-fold, respectively. This fact suggests that aldolase from rabbit muscle stimulates immunoglobulin production not only by the specified hybridoma cell line, but also by unspecified immunoglobulin producers; however, yeast aldolase was ineffective in accelerating immunoglobulin production by HB4C5 cells. The IPSF activity was lost by trypsin digestion at 100 μg ml −1; however, limited fragmentation by the lower concentrations of trypsin facilitated the IPSF activity of rabbit muscle aldolase about twofold even though the enzymatic activity was inactivated. These results imply that the immunoglobulin production-stimulating effect of aldolase is irrelevant to its enzymatic function, and the activity was defined as a novel role of rabbit muscle aldolase.

Spermine enhances IgM productivity of human-human hybridoma HB4C5 cells and human peripheral blood lymphocytes.

The polyamine spermine was assessed for enhancement of IgM production by human-human hybridoma, HB4C5 cells, under serum-free conditions. IgM production of HB4C5 cells was stimulated approximately 6-fold by the addition of 7.3 mM of spermine. The facilitating effect was observed soon after inoculation. In spite of suppression of cell growth, the IgM production rate was accelerated for at least 5 days without medium change. Moreover, laser confocal microscopic analysis revealed that the IgM content inside HB4C5 cells was increased by spermine treatment. These findings suggest that spermine enhances specific IgM productivity of the hybridoma line. Spermine also facilitated IgM production by human peripheral blood lymphocytes under serum-free conditions. This result implies that spermine enhances immunoglobulin production of not only specific hybridoma lines, but also non-specific immunoglobulin producers. Immunoglobulin production stimulating activity of spermine was accelerated 2-fold by the addition of DNA whith a chain length of about 400-7000 base pairs (bp). However, degraded short-chain DNA fragments (less than 200 bp) did not facilitate the immunoglobulin production stimulating activity of spermine.

Enhancement of production of IgM and interferon-beta in human cell lines by poly-lysine.

The promotive effects of poly-cations on immunoglobulin production was investigated using human-human hybridoma cells. Among poly-cations tested, epsilon-poly-L-lysine with hydrochloride (approximately 4 kDa), which has been used as an antibacterial food additive, had the greatest activity in enhancing IgM production of human-human hybridoma HB4C5 cells without stimulating cell proliferation. Immunoglobulin production stimulatory (IPS) activity of epsilon-poly-lysine was not affected by trypsin digestion. It was stable below 60 degrees C but completely inactivated with heating at 100 degrees C for 30 min. epsilon-Poly-lysine also enhanced interferon-beta (IFN-beta) production of human osteosarcoma MG-63 cells, but this stimulatory effect was reduced by the trypsin digestion.

Effects of organic pH buffers on a cell growth and an antibody production of human-human hybridoma HB4C5 cells in a serum-free culture

Human-human hybridoma cells secreting a human monoclonal antibody were cultured in a serum-free medium containing various organic pH buffers in order to clarify their effects on cell growth and antibody production. Organic pH buffers having either one sulfonic acid and several acyclic amine moieties, or several cyclic amine moieties containing two amino nitrogen did not inhibit cell growth; while other organic buffers sulfonic acid moiety plus several cyclic amine moieties containing one amino nitrogen slightly decreased cell growth, but enhanced antibody production. Using Fujita's organic conceptual diagram, a relationship between the organicity and inorganicity of a pH buffer to cell growth and antibody production was found. pH buffers with large inorganicity and small organicity values were favorable for cell growth, and buffers with small inorganicity and large organicity values were preferred to enhance antibody production. Although the pH buffering range affects cell growth, its effect on antibody production is not clear. In conclusion, 2-morpholinoethanesulfonic acid (MES), 3-morpholino-propanesulfonic acid (MOPS) and 1, 2-N, N'-bis[N″, N‴-di(2-sulfonoethyl)piperazinyl]ethane (Bis-PIPES) are shown to be the most optimal of the buffers tested, because they enhanced antibody production without decreasing the cell growth among the pH buffers tested here.

Stimulation of IgM production in human-human hybridoma HB4C5 cells by chitosan.

We screened for immunoglobulin production stimulating factors (IPSFs) in polysaccharides using human-human hybridoma cells, HB4C5, cultured in serum-free medium. Among polysaccharides, citrus pectin, locust bean gum, and chitosan stimulated IgM production of HB4C5 cells. Especially chitosan showed the strongest IPSF activity; 100 ng/ml of chitosan stimulated IgM production approximately 5-fold. Chitosan had several characteristics as IPSF, as follows. 1) For the IPSF activity, 70-90% deacetylation was essential. 2) Chitosan oligomers (n = 5, 6, 7) and chitin oligomers (n = 5, 6, 7) showed no IPSF activities. 3) The IPSF activity of chitosan was inhibited by glucosamine, one of the constitutive sugars of chitosan. 4) Chitosan stimulated IgM production of human lymphocytes in serum-free culture, but not IgG or IgA, nor in serum-supplemented culture.

Stimulation of Immunoglobulin Production in Human-human Hybridoma HB4C5 Cells and Human Lymphocytes by Soymilk.

Soymilk was found to stimulate IgM production in human-human hybridoma HB 4 C 5 cells cultured in serum-free ERDF medium supplemented with insulin (I), transferrin (T), ethanolamine (E) and selenine (S) (ITES-ERDF). The activity for immunoglobulin production stimulating factor (IPSF) was recognized in the acid precipitable fraction. The activity was observed in a peak obtained by fractionation on DEAE-Toyopearl 650M. Molecular size of the IPSF was estimened to be approximately 290kDa by gel filtration on Superose 12 HR. The susceptibility of the IPSF to heat or trypsin treatment suggested that it was a proteinous substance. Soymilk also stimulated IgM and IgG production in human lymphocytes cultured in ITES-ERDF medium.

Stimulation of IgM production in human-human hybridoma HB4C5 cells and human lymphocytes by soybean hull hemicellulose

Stimulation of IgM production in human-human hybridoma HB4C5 cells and human lymphocytes by soybean hull hemicellulose

Stimulation of proliferation and immunoglobulin production of human-human hybridoma by various types of caseins and their protease digests.

We have studied the effect of such milk proteins as caseins, lactalbumin, and lactoglobulin, on proliferation and immunoglobulin production of human-human hybridoma HB4C5 cells. It was found that alpha-, beta-, and kappa-caseins stimulated both proliferation and IgM product ion of human-human hybridoma HB4C5 cells, while the activities of alpha-lactalbumin and beta-lactoglobulin were negligible. To localize the active sites of these caseins, effect of protease treatments on the activities were examined. When caseins were digested with trypsin, casein digests stimulated proliferation of the hybridoma, but not their IgM production. When kappa-casein was digested with chymosin and fractionated to p-kappa-casein and glycomacropeptide, both fragments stimulated proliferation of the cells, but only p-kappa-casein fragment stimulated IgM production. These results indicate that kappa-casein has at least two proliferation stimulating sites and an IgM production stimulating site in the p-kappa-casein region.

Effects of phenyl compounds on proliferation and IgM production of human-human hybridoma HB4C5 cells cultured in serum-free medium.

Effects of phenyl compounds on proliferation and IgM production of human-human hybridoma HB4C5 cells cultured in serum-free medium.

Screening of immunoglobulin production stimulating factor (IPSF) in foodstuffs using human-human hybridoma HB4C5 cells.

We screened the immunoglobulin production stimulating factor (IPSF) in foodstuffs, using human-human hybridoma HB4C5 cells cultured in serum-free 1TES-ERDF medium, and found that egg yolk lipoprotein (YLP), lactoferrin, Block Ace, and casein had IPSF activity. The maximum IPSF activity was obtained at concentrations over 100^g/ml in YLP, 10μg/ml in lactoferrin, and 25 μg/ml in Block Ace and casein. These IPSFs stimulated the IgM production of human-human and mouse-mouse hybridomas, but their effect on IgG producers was very small. This suggests that IgG production of hybridomas is regulated differently from their IgM production.