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.     

Evaluation of immunostimulatory effect of the arrowroot (<Emphasis Type="Italic">Maranta arundinacea</Emphasis>. L) in vitro and in vivo

Arrowroot (Maranta arundinacea. L) is an underutilized local crop potentially to be developed as carbohydrate source and functional food in Indonesia. The objectives of this research are to evaluate the immunostimulatory effects of arrowroot extracts in vitro by using animal cell culture techniques, and in vivo by using BALB/c mice. The arrowroot tuber extracts were prepared by heat-treatment at 121 °C for 20 min in distilled water. The IgM production stimulatory activity of arrowroot tuber extracts against human hybridoma HB4C5 cells and mouse splenocytes was assessed. The result indicated that the arrowroot tuber extract stimulated IgM production by HB4C5 cells and immunoglobulin (IgG, IgA and IgM) production by splenocytes in vitro. In addition, the arrowroot tuber extracts strongly enhanced interferon γ production by splenocytes. In vivo study indicated that the diet containing arrowroot extracts increased the serum IgG, IgA and IgM levels in mice. These results revealed that the arrowroot tuber extracts have immunostimulatory effects in vivo as well as in vitro.     

Lysozyme stimulates immunoglobulin production by human-human hybridoma and human peripheral blood lymphocytes

Lysozyme [EC 3.2.1.17] derived from hen egg white stimulated immunoglobulin production by human-human hybridoma, HB4C5 cells producing human lung cancer specific monoclonal IgM. IgM production by HB4C5 cells was enhanced more than 13-fold by the addition of lysozyme at 380 μg/ml in a serum-free medium. The immunoglobulin production stimulating effect of lysozyme was observed immediately after inoculation and maintained for 5 days. Lysozyme enhanced immunoglobulin production by the hybridoma line without growth promotion. This enzyme also accelerated IgM and IgG production of human peripheral blood lymphocytes 5.3-fold and 2.3-fold, respectively. These results suggest that lysozyme stimulates immunoglobuling production of not only specific hybridoma line, but also non-specific immunoglobulin producers. However, although the enzymatic activity of lysozyme was almost lost by heat-treatment at 100 °C for 30 min, the IPSF activity was retained. This fact suggests that IPSF activity of lysozyme does not come from its enzymatic activity or reaction products. All these findings clearly indicate that lysozyme has a novel function as an immunoglobulin production stimulating factor. GAPDH - glyceraldehyde-3-phosphate dehydrogenase; Ig - immunoglobulin; IPSF - immunoglobulin production stimulating factor; PBL - peripheral blood lymphocytes; HPLC - high-performance liquid chromatography. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

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.     

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.     

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.     

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.     

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.     

Immunomodulatory activity of Bengkoang (Pachyrhizus erosus) fiber extract in vitro and in vivo

Bengkoang (Pachyrhizus erosus (L.) Urban) is one of the most popular edible root vegetables in Indonesia. Bengkoang contains fairly large amounts of carbohydrates and crude fiber. The purpose of this research is to evaluate the immunomodulatory effect of the bengkoang fiber extract (BFE) in vitro and in vivo. BFE was prepared by heating the powder of bengkoang fiber suspended in distilled water at 121 °C for 20 min. BFE facilitated IgM production by the human hybridoma cell line HB4C5 cells. In addition, production of IgM, IgG, and IgA by mouse primary splenocytes was facilitated by BFE in a dose-dependent manner. BFE also significantly facilitated production of both interleukin-5 and interleukin-10 by splenocytes. Immunoglobulin production by lymphocytes from the spleen, Peyer’s patch, and mesenteric lymph node were significantly activated by oral administration of BFE to mice for 14 days. The serum immunoglobulin levels of IgG, IgM, and IgA were also significantly enhanced. Furthermore, cytokine production by lymphocytes from the spleen, Peyer’s patch, and mesenteric lymph node were also facilitated by oral administration of BFE. These results suggest that BFE has positive effects on the immune system in vitro and in vivo.     

Effect of some constituents of chicken egg yolk lipoprotein on the growth and IgM production of human-human hybridoma cells and other human-derived cells

Chicken egg yolk lipoprotein (YLP) was partially fractionated into some constituents, and the effect of constituents of YLP were examined on the growth and immunoglobulin (IgM and IgG) secretion of a HB4C5 human-human hybridoma cell line cultured in serum-free medium. Among the fractions, YP-1 and YP-2 fractions (LDL-rich fractions) were found to enhance the growth and IgM secretion of HB4C5 cells. The promoting activity was found in the commercial LDL. The lipid fraction in YP-2 fraction conjugated with 2-maltosyl-a-cyclodextrin was found to enhance the growth and IgM secretion of HB4C5 cells. Livetin-rich YP-3 and YP-4 fractions had no significant promoting activity. Commercial -livetin and phosphatidyl choline possessed no growth-promoting activity. Phosphatidyl choline enhanced the IgM secretion of HB4C5 cells.     

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.     

Stimulation of proliferation and immunoglobulin M production by lactoferrin in human-human and mouse-mouse hybridomas cultures in serum-free conditions

The effects of growth factors, such as insulin, transferrin, lactoferrin, ethanolamine, and selenium, on proliferation and IgM production of human-human hybridomas HB4C5 cells in a serum-free enriched RDF (eRDF) medium were studied. Among them, lactoferrin markedly stimulated proliferation and IgM production of the cells. Another iron-binding protein, transferrin, stimulated proliferation of HB4C5 cells as well as lactoferrin, but its stimulatory effect on IgM production was negligible. The proliferation and IgM production of HB4C5 cells gave some detectable delays in conventional ITES-eRDF medium at low cell densities, but the delays were avoided by the addition of lactoferrin. However, eRDF medium supplemented with lactoferrin could not support proliferation and IgM production of the cells at high cell densities. For proliferation and IgM production of HB4C5 cells, eRDF medium supplemented with 25 g/ml lactoferrin, 10M ethanolamine, 35 /ml transferrin, and 2.5 nM selenium (LETS-eRDF) gave the best result. Lactoferrin stimulated proliferation of human-human and mouse-mouse hybridomas producing IgG or IgM, but stimulation of Ig production was detected only in IgM producers. These results suggest that cell proliferation, IgM production, and IgG production of hybridomas are regulated by different mechanisms.Abbreviations MoAb monoclonal antibody     

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.     

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.     

Immunostimulatory in vitro and in vivo effects of a water-soluble extract from kale

The water-soluble fraction of kale (Brassica oleracea L. var. acephala DC.) had immunoglobulin (Ig) production stimulating activity in human hybridoma HB4C5 cells and human peripheral blood lymphocytes. The biochemical and physical properties of the main active substance in kale were found to be a heat-stable protein with a molecular weight higher than 50 kDa. The Ig production-stimulating factors were assumed to act on the translational and/or secreting processes of Igs. This Ig production-stimulating effect was also observed in lymphocytes from the mesenteric lymph node and Peyer's patches of mice that had been administered with the kale extract for 14 d. The partially purified kale extract was analyzed by LC-ESI-MS/MS, the result indicating ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) as an active substance. Rubisco from spinach indeed exhibited Ig production-stimulating activity in HB4C5 cells. These findings provide another beneficial aspect of kale as a health-promoting foodstuff.