Yoshifumi Tomita

Immunomodulating actions of carotenoids: Enhancement of in vivo and in vitro antibody production to T‐dependent antigens

Previously, we demonstrated an enhancement of in vitro antibody (Ab) production in response to T-dependent antigens (TD-Ag) by astaxanthin, a carotenoid without vitamin A activity. The effects of beta-carotene, a carotenoid with vitamin A activity, and lutein, another carotenoid without vitamin A activity, on in vitro Ab production were examined with spleen cells from young and old B6 mice. In addition, the in vivo effects of lutein, astaxanthin, and beta-carotene on Ab production were studied in young and old B6 mice. Lutein, but not beta-carotene, enhanced in vitro Ab production in response to TD-Ags. The depletion of T-helper cells prevented the enhancement of Ab production by lutein and astaxanthin. In vivo Ab production in response to TD-Ag was significantly enhanced by lutein, astaxanthin, and beta-carotene. The numbers of immunoglobulin M- and G-secreting cells also increased in vivo with the administration of these carotenoids when mice were primed with TD-Ags. Antibody production in response to TD-Ags in vivo and in vitro was significantly lower in old than in young B6 mice. Astaxanthin supplements partially restored decreased in vivo Ab production in response to TD-Ags in old B6 mice. Lutein and beta-carotene also enhanced in vivo Ab production in response to TD-Ags in old B6 mice, although to a lesser extent than did astaxanthin. However, none of the carotenoids had an effect on in vivo or in vitro Ab production in response to T-independent antigen. These results indicate significant immunomodulating actions of carotenoids for humoral immune responses to TD-Ags and suggest that carotenoid supplementation may be beneficial in restoring humoral immune responses in older animals.

Studies of immunomodulating actions of carotenoids. II; Astaxanthin enhances in vitro antibody production to T-dependent antigens without facilitating polyclonal B-cell activation

Previously we have shown that astaxanthin, a carotenoid without provitamin A activity, enhances in vitro antibody (Ab) production to sheep red blood cells in normal B6 mice. In this study, we further attempted to examine the mechanisms of this enhancing action of carotenoids on specific Ab production in vitro in relation to different antigen (Ag) stimuli, cytokine production, and T- and B-cell interactions in both normal and autoimmune strains of mice. When the actions of carotenoids were tested in normal strains of mice, we found that astaxanthin enhanced in vitro Ab production to T cell-dependent Ag, but not to T-independent Ag, and did not augment total immunoglobulin production. Astaxanthin exerted maximum enhancing actions when it was present at the initial period of Ag priming. This action of astaxanthin was abolished when T cells were depleted from spleen cell suspensions and appeared to require direct interactions between T and B cells. The results also indicated that carotenoids may modulate the production of interferon-tau in this assay system. When the actions of carotenoids were tested in autoimmune-prone MRL and NZB mice, the enhancing action of astaxanthin on in vitro Ab production was less significant. Furthermore, carotenoids did not potentiate or augment spontaneous Ab and immunoglobulin production by spleen cells in these strains. Taken together, carotenoids without provitamin A activity may be able to augment in vitro specific Ab production to T cell-dependent Ag partly through affecting the initial stage of Ag presentation without facilitating polyclonal B-cell activation or autoantibody production.

Studies of immunomodulating actions of carotenoids. I. Effects of β‐carotene and astaxanthin on murine lymphocyte functions and cell surface marker expression in in vitro culture system

The immunomodulating effects of carotenoids (beta-carotene and astaxanthin) on mouse lymphocytes were studied in in vitro culture system by use of assay for mitogen responses of spleen cells, thymocyte proliferation, interleukin 2 production, and antibody (Ab) production in vitro in response to sheep red blood cells. Changes of cell surface markers on spleen lymphocytes including Ia antigen (Ag), surface immunoglobulin, B220, and Thy-1 Ag were also examined. At a concentration of 10(-8) M, carotenoids did not show any significant effect on mitogen responses (phytohemagglutinin P and concanavalin A) on murine spleen cells, irrespective of the concentrations of mitogens used. Interleukin 2 production by murine spleen cells was not significantly altered by carotenoids in the culture media (10(-7) to 10(-9) M). [3H]thymidine incorporation by B6 thymocytes was somewhat enhanced in the presence of astaxanthin or beta-carotene when cultured in the concentration of 10(6)/ml. At higher concentrations of cells (5 x 10(6)/ml), such an effect was not observed. In assays of in vitro Ab production in response to sheep red blood cells, B6 spleen cells produced significantly more Ab-forming cells (plaque-forming cells, immunoglobulins M and G) in the presence of astaxanthin (greater than 10(-8) M) but not beta-carotene. Expression of Ia Ag seemed to be moderately enhanced on both Thy-1+ and Thy-1- spleen cells in the presence of astaxanthin (greater than 10(-9) M) but not beta-carotene. The expression of Thy-1 and surface immunoglobulin seemed unchanged with the treatment of these carotenoids. These results indicate that immunomodulating actions of carotenoids are not necessarily related to provitamin A activity, because astaxanthin, which does not have provitamin A activity, showed more significant effects in these bioassays and also indicate that such actions of carotenoid demonstrated in this study may be difficult to explain only by its oxygen-quenching capacity.

Oral Administration of Chlorella Vulgaris Augments Concomitant Antitumor Immunity

Chlorella vulgaris, an unicellular green algae, or its acetone-extract (Ac-Ex) were administered orally to Meth A tumor bearing BALB/c or (BALB/c x DBA/2)F1 (CDF1) mice. When CDF1 mice were fed daily with 10% dried powder of Chlorella vulgaris (CVP) containing diet before and after Meth A tumor inoculation, the growth of rechallenged Meth A tumor was significantly suppressed in an antigen-specific manner. Augmentation of antitumor resistance was exhibited also by Winn assay using lymph node cells of tumor-bearing mice orally administered with CVP or Ac-Ex. Antigen-specific concomitant immunity in these mice were mediated by cytostatic T cells but not by cytotoxic T cells. Natural killer cells seemed not to contribute in antitumor resistance in this system.

Immunological role of vitamin A and its related substances in prevention of cancer.

The antitumor effects of vitamin A and its related substances, vitamin E, vitamin K, beta-carotene, ubiquinone, phytol, and squalene, were examined using a syngeneic murine tumor system. Intraperitoneal administration of these substances (0.19 mumol/mouse/day) slightly suppressed the growth of Meth A fibrosarcoma cells inoculated s.c. into Balb/c mice. Administration of all test substances except beta-carotene significantly suppressed the growth of Meth A fibrosarcoma cells rechallenged in Meth A-primed mice on day 10, but did not influence the growth of Meth 1 fibrosarcoma cells (another syngeneic tumor of Balb/c origin) rechallenged in Meth A-primed mice on day 10. The growth of Meth A tumor cells was suppressed when Meth A was inoculated together with lymph node cells obtained from the Meth A-primed Balb/c mice treated with vitamin A, vitamin E, phytol, or squalene. Our findings suggest that certain constituents in green-yellow vegetables may contribute to the prevention of cancer by augmenting an immunological response against tumor cells in the early stages of carcinogenesis.

PREVENTIVE ACTION OF CAROTENOIDS ON THE DEVELOPMENT OF LYMPHADENOPATHY AND PROTEINURIA IN MRL-lpr/lpr MICE

The chemopreventive action of carotenoids on proteinuria and lymphadenopathy were examined in autoimmune-prone MRL-lpr/lpr (MRL/l) mice. They were fed a synthetic full-fed diet (16-18 kcal/mouse/day) with supplementation of beta-carotene or astaxanthin (0.19 mumoles/mouse, 3 times a week), and the development of lymphadenopathy and proteinuria were examined. MRL/l mice fed a full-fed diet without the supplementation of carotenoids or those fed a calorie-restricted (CR) diet (10-11 kcal/mouse/day, 60% calorie intake of full-fed mice) were employed as controls. CR dramatically delayed the development of proteinuria and lymphadenopathy, as reported previously. Carotenoids also significantly delayed the onset of these symptoms in MRL/l mice fed a full-fed diet. Carotenoids were half as effective as CR and astaxanthin, a carotenoid without provitamin A activity, which appeared to exert more significant preventive actions than beta-carotene in delaying the development of these symptoms. Similar chemopreventive actions of carotenoids were also demonstrated in MRL/l mice fed a regular diet (Lab Chow). CR has been shown to augment IL-2 production and to decrease serum prolactin levels in this strain, which may be related to its dramatic preventive action of autoimmunity. However, carotenoids did not affect IL-2 production nor prolactin levels in full-fed MRL/l mice. The chemopreventive actions of carotenoids observed in autoimmune-prone MRL/l mice may be attributed to yet unknown mechanisms, apart from their provitamin A activity or oxygen-quenching activity.

Vitamin A and tumor immunity

Intraperitoneal administration of vitamin A into the BALB/c mice inoculated with a syngeneic fibrosarcoma, Meth A, caused a remarkable augmentation of tumor rejection. A cell-depletion technique revealed that the primary effector cells responsible for the augmented rejection were Thy-1 positive, Lyt-1 negative, Lyt-2 positive lymphocytes, suggesting the involvement of cytotoxic lymphocytes.

Inhibitory Effect of Gramicidin S on the Growth of Murine Tumor Cells in vitro and in vivo

The Effect of gramicidin S (GS), a polypeptide antibiotic, on the growth of murine tumor cells such as allotransplantable sarcoma 180 (S180) and Ehrlich ascites carcinoma (EAC) and Meth A fibrosarcoma (Meth A) was studied. GS inhibited the proliferation of EAC cells in culture and its effect on cell viability was dependent on the concentration of GS. On exposure to GS at concentrations ranging from 1 to 100 micrograms/ml for 10 min, EAC cells lost their transplantability in ddY mice depending on the concentration of GS. In particular, the transplantability of EAC cells was completely missing on exposure to GS at a concentration of 100 micrograms/ml. In the in vivo experiments, a daily intraperitoneal injection of GS exhibited a high inhibitory effect not only on the growth of subcutaneously implanted S180 in ICR mice but on the growth of subcutaneously implanted syngeneic Meth A in BALB/c mice. In the studies using radioactively labeled DNA, RNA and protein precursors, GS at high concentrations inhibited the incorporation of all the precursors into EAC cells.

Dietary survey methods in analytic epidemiology.

慢性疾患の分析疫学的研究に資する目的で, 種々の食習慣調査方法が開発され工夫されてきたが, 潜伏期間が長いこと, 個体差が大きいこと, 食習慣が多面にわたることなどのため, 実用的で妥当で再現性の高い方法を特定するのはむずかしい。食習慣の多様化と年次的変化は食習慣と疾患との関係をいっそう複雑なものにしており, 食習慣調査方法とその性能に関して今後の研究が大いに望まれる分野である。