Heat denaturation enhanced immunoglobulin production stimulating activity of lysozyme from hen egg white

Lysozyme from hen egg white was identified as an immunoglobulin production stimulating factor (IPSF) that enhances immunoglobulin production by hybridomas and lymphocytes. The IPSF activity of lysozyme was facilitated by heat treatment. The heat treatment of lysozyme at 83 degrees C for 30 min activated its specific IPSF effect 30.0-fold compared with that of native lysozyme. The IPSF activity of lysozyme heat-treated at 83 degrees C in 4 M urea solution was enhanced 8.4-fold than that of native lysozyme. However, lysozyme that was not heated in 4 M urea solution completely lost its IPSF activity. This means that the IPSF activity of this enzyme in 4 M urea was reactivated by thermal treatment. Moreover, coexistence of 0.5 mM 2-mercaptoethanol (2-ME) during heating in 4 M urea solution extremely enhanced the IPSF activity up to 77.8-fold. The uptake of lysozyme by hybridoma cells was enhanced by heat denaturation in 4 M urea. The hydrophobicity of lysozyme was extremely increased by heat-treatment in 2-ME containing urea solution. It is expected from these findings that the increase in the hydrophobicity caused the enhancement of incorporation of lysozyme into target cells, and resulted in the acceleration of IgM production.     

Alcohol dehydrogenase-I from horse liver stimulates immunoglobulin production by human hybridoma and human peripheral blood lymphocytes

Immunoglobulin production stimulating activity of alcohol dehydrogenase[EC 1.1.1.1] was assessed. Alcohol dehydrogenase-I (ADH-I) derived fromhorse liver stimulated IgM production by human-human hybridoma, HB4C5 cellsproducing human lung cancer specific monoclonal IgM. IgM production of HB4C5cells was enhanced more than 6 fold by the addition of ADH-I at 400µg/ml under serum-free condition. However, yeast derived ADHs, such asADH-II and -III were ineffective to accelerate immunoglobulin production ofthe hybridoma line. These results imply that the immunoglobulin productionstimulating effect of ADH-I is irrelevant to its enzymatic function, anddefined as a novel feature of ADH-I. This enzyme also stimulated IgM and IgGproduction by human peripheral blood lymphocytes 2.9 fold and 1.4 fold,respectively . This fact suggests that ADH-I stimulates immunoglobulinproduction not only by specific hybridoma cell line, but also bynon-specific immunoglobulin producers.     

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.     

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. This revised version was published online in August 2006 with corrections to the Cover Date.     

Immunoglobulin production stimulating and inhibiting factors derived from human lung adenocarcinoma PC-8 cells

Addition of a 3 M KCl extract of a human lung adenocarcinoma PC-8 cells to the culture media of lymphocytes, which were isolated from normal donors and from lung or breast cancer patients, elevated immunoglobulin (Ig) production by 3–5 times in the presence of pokeweed mitogen. However, addition of higher concentration of the extract inhibited Ig production and proliferation of lymphocytes. The Ig production stimulating factor (IPSF) was separated from the inhibiting factor, using 50% ammonium sulfate precipitation. Upon chromatofocusing, IPSF activity was detected mainly in the pH 4.5 fraction but minor activity was also detected in other pH fractions. IPSF also enhanced Ig production of a B-lymphoblastoid cell line transformed by Epstein-Barr virus and a human-human hybridoma, by more than 2-fold. This suggests that IPSF interacts with B-lymphocytes directly to enhance their Ig production. IPSF activity was also detected diversely in human lung squamous carcinoma QG56, human B-lymphoblastoid HO-323, and a T cell line CEM.     

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.     

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.     

Effects of polyamines on proliferation and IgM productivity of human–human hybridoma,HB4C5 cells

Immunoglobulin production stimulating activity of polyamines was investigated. Spermidine, thermine and triethylenetetraamine (TETA) stimulated IgM production of human–human hybridoma, HB4C5 cells under serum-free condition. IgM production of HB4C5 cells was accelerated 5.9-, 5.3-, and 3.7-fold by spermidine at 4.5 mM, thermine at 2 mM and TETA at 2.5 mM, respectively. However, putrescine did not enhance IgM production. Spermidine enhanced IgM productivity of the hybridoma cells in spite of its growth suppression activity. TETA also inhibited cell proliferation and the effect on the acceleration of IgM productivity disappeared during 5 days because of its cytotoxicity. On the other hand, thermine facilitated IgM productivity of the hybridoma cells without growth suppression. The laser confocal microscopic analysis revealed that IgM content inside HB4C5 cells was increased by thermine. This result suggests that thermine facilitates IgM synthesis in hybridoma cells.     

Constituents of Japanese Peppermint Oil

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121 °C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20 d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.     

Effects of DNA on immunoglobulin production stimulating activity of alcohol dehydrogenase

Alcohol dehydrogenase-I (ADH-I) derived from horse liver stimulated IgM production by human-human hybridoma, HB4C5 cells and lymphocytes. The IPSF activity of ADH-I was suppressed by coexistence of short DNA whose chain length is less than 200 base pairs (bp) and fibrous DNA in a dose-dependent manner. These DNA preparations completely inhibited the IPSF activity at the concentration of 250 μg/ml and 1.0 mg/ml, respectively. DNA sample termed long DNA whose average chain length is 400–7000 bp slightly stimulated IPSF activity at 0.06 μg/ml. However, long DNA suppressed IPSF activity by half at 1.0 mg/ml. The laser confocal microscopic analysis had revealed that ADH-I was incorporated by HB4C5 cells. The uptake of ADH-I was strongly inhibited by short DNA and fibrous DNA. However, long DNA did not suppress the internalization of ADH-I into HB4C5 cells. These findings indicate that short DNA and fibrous DNA depress IPSF activity of ADH-I by inhibiting the internalization of this enzyme. According to the gel-filtration analysis using HPLC, ADH-I did not directly interact with short DNA. It is expected from these findings that short DNA influences HB4C5 cells to suppress the internalization of ADH-I. Moreover, these facts also strongly suggest that ADH-I acts as IPSF after internalization into the cell. This revised version was published online in August 2006 with corrections to the Cover Date.     

Partial purification and characterization of immunoglobulin production stimulating factor derived from namalwa cells

Summary We screened for immunoglobulin (Ig) production stimulating factor (IPSF) which enhanced Ig production of human-to-human hybridomas in serum-free culture, and found that culture supernatant and lysate of human lymphoblastoid Namalwa cells stimulated proliferation and Ig production of human-to-human hybridoma HB4C5 cells. The IPSF in Namalwa lysate was partially purified with DEAE-Toyopearl 650M, hydroxylapatite and Superose 6HR 10/30 column chromatographies. The partially purified IPSF was a macromolecule of about 500 000 dalton containing 72 000 dalton protein as a major component. The activity was stable at pH 6 to 12, but inactivated partially by heating over 40° C (60% decrease) and completely by trypsin digestion. These results suggest that the IPSF activity is due to its protein and heat-stable components. The Namalwa IPSF stimulated proliferation of human-to-human hybridomas but not that of mouse-to-mouse hybridomas. The IPSF also stimulated Ig production of human-to-human hybridomas derived from NAT-30 cells, but not that of other human-to-human or mouse-to-mouse hybridomas. NAT-30 is a human fusion partner derived from Namalwa cells. These results suggest that the Namalwa IPSF is an autocrine factor that stimulates proliferation and Ig production of hybridomas derived from NAT-30 cells. This work was supported in part by a grant-in-aid from the Ministry of Education, Science and Culture (Japan) and by Sapporo Bioscience Foundation.     

Isolation of Escherichia coli B Mutant Deficient in Glutathione Biosynthesis

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 °C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-γ and tumor necrosis factor (TNF)-α by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles’ tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Purification and characterization of immunoglobulin production stimulating factor derived from human B lymphoblastoid HO-323 cells

An immunoglobulin (Ig) production stimulating factor (IPSF) for hybridomas was found in spent medium of the human B lymphoblastoid cell line, HO-323. The IPSF was purified by serial use of DEAE chromatography, ultrafiltration, gel filtration and HPLC-DEAE chromatography. Purified IPSF was estimated to be a 410 k macro molecule by gel filtration, and contained three types of isomers which were separated from each other by native polyacrylamide gel electrophoresis. All of the isomers were, however, assumed to have the same protein components by SDS polyacrylamide gel electrophoresis.The IPSF was effective for human-human and mouse-mouse hybridomas producing IgM, but not for IgG producers in the experimental condition used here. Human-human hybridoma HF10B4, cultured in IPSF-containing medium, produced 20 times more IgM than in IPSF-free medium under serum-free conditions. The IPSF showed very little proliferation stimulating activity on HF10B4 cells.     

Immunostimulation effects of yellowtail heart extracts in vitro and in vivo

The immunostimulation effects of yellowtail heart extracts were examined. Screening various parts of the yellowtail viscera, we found that extracts from the yellowtail heart enhanced IgM production by human hybridoma HB4C5 cells. Yellowtail heart extracts heated at 121°C for 20 min and dialyzed showed the highest IgM production-stimulating activity toward HB4C5 cells. Also, immunoglobulin production by mouse spleen lymphocytes was stimulated by yellowtail heart extracts in vitro, and lymphocytes derived from mice administered the extract for 20 d were activated in vivo. Yellowtail heart extracts were partially purified by anion-exchange chromatography, and fractions containing a 33 kDa-protein exhibited immunostimulating activity. LC-MS/MS analysis revealed that the 33 kDa-protein was most similar to tropomyosin-4 from various fishes. Purified tropomyosin from porcine muscle enhanced IgM production by HB4C5 cells. This means that tropomyosin-4 is one of the immunostimulating substances in the yellowtail heart.     

Immunostimulation effect of jellyfish collagen

Certain edible large jellyfishes belonging to the order Rhizostomeae are consumed in large quantities in China and Japan. The exumbrella part of the edible jellyfish Stomolophus nomurai was cut and soaked in dilute hydrochloric acid solution (pH 3.0) for 12 h, and heated at 121 degrees C for 20 min. The immunostimulation effects of the jellyfish extract were examined. The jellyfish extract enhanced IgM production of human hybridoma HB4C5 cells 34-fold. IgM and IgG production of human peripheral blood lymphocytes (PBL) were also accelerated, 2.8- and 1.4-fold respectively. Moreover, production of interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha by human PBL was stimulated 100- and 17-fold respectively. Collagenase treatment inactivated the immunostimulation activity of the jellyfish extract. In addition, purified collagen from bovine Achilles' tendon accelerated IgM production of hybridoma cells. These facts mean that collagen has an immunostimulation effect, and that the active substance in jellyfish extract is collagen.     

Immunostimulation effects of proteose-peptone component 3 fragment on human hybridomas and peripheral blood lymphocytes

Fat-free bovine milk fermented by 12 kinds of lactic acid bacteria and yeast enhanced monoclonal antibody production of human hybridoma HB4C5 cells 2.8-fold in serum-free medium. Immunoglobulin production of human peripheral blood lymphocytes (PBL) was also stimulated in vitro. IgM and IgG production of human PBL was accelerated up to 2.8-fold and 5.4-fold, respectively. Interferon-gamma production of human PBL was also accelerated 6.0-fold by 50 microg/ml of the fermented milk. However, interleukin-4 production of PBL was not affected, and tumor necrosis factor-alpha production was suppressed. The activity was enhanced 2.5-fold by the thermal treatment for 30 min at 65 degrees C and was completely lost by trypsin digestion. The findings suggested that the active substance in the fermented milk was heat stable protein. Gel-filtration and the SDS-PAGE analysis revealed that the molecular weight of the active substance was estimated as 19.0 kDa, which was not detected in fat-free bovine milk before fermentation. N-terminal amino acid sequence of the 19.0 kDa protein was highly homologous to proteose-peptone component 3 (PP3). Since molecular weight of PP3 is 28 kDa, it is suggested that the 19.0 kDa protein is derived from degradation of PP3 during fermentation of fat-free milk. Moreover, PP3 purified from fat-free milk also enhanced IgM production of HB4C5 cells.