The Journal of Nutrition Vol. 128 No. 2 February 1998, pp. 193-197

Dietary Supplementation with Mushroom-Derived Protein-Bound Glucan Does Not Enhance Immune Function in Young and Old Mice^1,2

Dayong Wu, Sung Nim Han, Roderick T. Bronson, Donald E. Smith, and Simin Nikbin Meydani³

Nutritional Immunology Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA 02111

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Decline in immune response is a well-documented age-associated biological change. Protein-bound polysaccharides (PSP) are biological response modifiers and have been shown to have immunoenhancing and antitumor effects. This study was conducted to examine the effect of dietary supplementation with PSP-containing extract derived from mycelia of Coriolus versicolor on in vitro and in vivo indices of immune function of young and old mice. Young (5 mo) and old (23 mo) C57BL/6NIA mice were fed purified diets containing 0, 0.1, 0.5 or 1.0% PSP for 1 mo at which time indices of immune function were measured. PSP supplementation had no significant effect on mitogenic response to concanavalin A (Con A), phytohemagglutinin (PHA) or lipopolysaccharide (LPS), or on production of interleukin (IL)-1, IL-2, IL- 4 and prostaglandin E₂ (PGE₂). Of the in vivo indices of immune function tested, old mice fed 1.0% PSP had significantly higher delayed-type hypersensitivity (DTH) response than those fed 0% PSP. No significant effect of PSP was observed on the DTH response of young mice. The antibody response to sheep red blood cells was not significantly influenced by PSP in young or old mice. These results suggest that PSP-containing extract from mycelia of Coriolus versicolor might have a modest immunoenhancing effect in aged mice, but not in young mice.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Age-related decline in immune response has been well documented in numerous animal and human studies. Nutritional interventions have demonstrated promising potential in modulating the dysregulated immune response observed with age and certain pathologic conditions. For example, dietary supplementation with antioxidants such as vitamin E and glutathione or with multivitamin and mineral mixtures have been shown to improve immune function in aged mice and humans (Bogden et al. 1994, Chandra 1992, Furukawa et al. 1987, Meydani et al. 1990).

Protein-bound polysaccharides, or polysaccharide peptides (PSP),⁴ are a class of compounds found in abundance in certain mushrooms. They have been widely used as biological response modifiers (BRM) in Asian countries (Jong and Birmingham 1993, Matsunaga et al. 1987 and 1992b, Yang et al. 1993). PSP has been claimed to enhance immune function, inhibit cancer growth and metastasis and to increase host resistance to bacterial, viral and parasitic infections (Jong and Birmingham 1993). It has been postulated that the antitumor activity of PSP is an indirect enhancement of immune response rather than a direct cytotoxic effect on tumor cells (Jong and Birmingham 1993). Mushroom extracts containing PSP are widely sold as oral supplements in health food stores as antiaging, immunoenhancing or antitumor products. There are, however, few published, well-controlled studies of the effect of PSP on the immune response, particularly in unchallenged and healthy hosts, to substantiate the immunostimulatory claims attributed to these compounds. In addition, the few studies available have often used intraperitoneal injections as the route of administration (Hamuro et al. 1994, Matsunaga et al. 1992b, Penna et al. 1996), whereas the products available to the public are for oral use. Because the aged have decreased immune function and are at higher risk for infection and cancer, we examined the effect of dietary supplementation with a mushroom extract containing PSP on in vitro and in vivo immune function of old and young mice.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals and diets.  Specific pathogen-free male young (5 mo) and old (23 mo) C57BL/6NIA mice were obtained from National Institute on Aging colony at Charles River Laboratories (Kingston, NY). The mean life span of these mice is 28 mo. Mice were individually housed in microisolator cages maintained at a constant temperature (23°C) with a 12-h light:dark cycle. All conditions and handling of the animals were approved by the Animal Care and Use Committee at the Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University and conducted according to the NIH Guidelines for the Care and Use of Laboratory Animals.

For the in vitro study of cell-mediated immune response, 64 young and 64 old mice were evenly divided into four groups and fed purified diets supplemented with 0, 0.1, 0.5 or 1.0% extract of the mycelia of Coriolus versicolor (gift from Winsor Health Products, Hong Kong) for 1 mo. The composition of the purified diet is shown in Table 1. The PSP product contains 40-50% glucan, 20-30% protein, 11-33% minerals, 2-3% fat and 5-6% water, thus containing 60-80% PSP in form of protein-bound glucan. The PSP product (in form of powder) was mixed with basal diet by using a Hobart mixer (Hobart, Troy, OH) for 20 min to assure an even distribution of PSP in experimental diets. The diets, therefore, provided from 0 to 857 mg/(kg body weight·d) of protein-bound glucan to each mouse. The recommended dose by the manufacturer of this product (3 times daily, 1-3 capsules each time) will provide 17-51 mg/kg body weight for a human weighing 70 kg. Other PSP-containing products sold in nutrition stores suggest similar or smaller amounts of equivalent ingredients. For the study of in vivo cell-mediated immune response [delayed-type hypersensitivity, (DTH)] and T cell-dependent humoral immune response [antibody titer against sheep red blood cells (SRBC)], 40 young and 40 old mice were divided into two groups and fed the diets containing 0 or 1% PSP for 1 mo. Ten mice per treatment were used for DTH measurement and the other 10 for antibody measurement. Fresh food was provided daily. At the end of study, mice were paired by age and killed via CO₂ asphyxiation. Hearts, lungs, kidneys, pancreases, salivary glands and submandibular lymph nodes were removed for pathological examination. Mice exhibiting visible tumors or splenomegaly were excluded from the study.

Splenocyte isolation.  Spleens were aseptically removed and placed in sterile RPMI 1640 (BioWhitaker, Walkerville, MD) media supplemented with 25 mmol HEPES/L (Gibco, Grand Island, NY), 2 mmol glutamine/L (Gibco), 1 × 10⁵ units penicillin/L and 100 mg streptomycin/L (Gibco). Media supplemented as described will be referred to as complete RPMI. Single cell suspensions were prepared by gently disrupting spleens between two sterile frosted glass slides. Splenocytes were isolated via centrifugation (400 × g), and red cells were lysed using Grey's reagent. Splenocytes were washed twice with complete RPMI and viability was determined by trypan blue exclusion. Splenocytes were suspended in complete RPMI containing 10% heat-inactivated fetal bovine serum (Gibco) at appropriate concentrations for different cultures.

Mitogenic response.  Splenocytes were added (1 × 10⁵ cells/well) to 96-well flat bottom plates (Becton Dickinson Labware, Lincoln Park, NJ) and cultured in the presence or absence of the T cell mitogens concanavalin A (Con A; Sigma, St Louis, MO) at 0.5, 1.5 and 5 mg/L or phytohemagglutinin (PHA; Difco Laboratories, Detroit, MI) at 5, 20 and 40 mg/L, or the B cell mitogen lipopolysaccharide (LPS; Sigma) at 5, 15 and 30 mg/L for 72 h at 37°C in an atmosphere of 5% CO₂ and 95% humidity. Cultures were pulsed with 18.5 µBq [³H]-thymidine (DuPont NEN Products, Boston, MA) during the final 4 h of incubation. The cells were harvested onto glass fiber filter mats (Wallac, Gaithersburg, MD) by Tomtec harvester (Wallac), and cell proliferation was quantified as the amount of [³H]-thymidine incorporation into DNA as determined by liquid scintillation counting in a 1205 Betaplate counter (Wallac). The counter had an efficiency of >50% for ³H. Data are expressed as corrected counts per minute (ccpm) which is the cpm of mitogen-stimulated cultures minus the cpm of cultures without mitogen.

Cytokine production.  For interleukin (IL)-1 production, splenocytes were cultured at 5 × 10⁶ cells/well in the presence of LPS (100 mg/L) in 24-well culture plates (Becton Dickinson) for 24 h. For IL-2 and IL-4 production, splenocytes were cultured at 1 × 10⁶ cells/well in the presence of Con A (10 mg/L) and PHA (20 mg/L) in 96-well flat bottom plates (Becton Dickinson) for 24 h. Cell-free supernatants were collected and stored at 20°C for later analysis. IL-1, IL-2 and IL-4 were measured by using ELISA. The IL-1 ELISA kit was purchased from Genzyme (Cambridge, MA); recombinant IL-2, IL-4 and capture and detection antibodies of IL-2 and IL-4 were purchased from PharMingen (San Diego, CA). ELISA were conducted using a sandwich method described in the manufacturers' protocols.

Prostaglandin (PG) E₂ production.  Splenocytes were cultured at 5 × 10⁶ cells/well in the presence of Con A (5 mg/L) in 24-well culture plates (Becton Dickinson) for 48 h. Cell-free supernatants were collected and stored at 70°C for later analysis. PGE₂ was measured by RIA as described by McCosh et al. (1976). PGE₂ antibody was a gift from J. Dupont of Iowa State University and M. Mathias of Colorado State University. The antibody specificity and cross-activity were described previously (Meydani and Dupont 1982).

Delayed-type hypersensitivity.  DTH was measured by a radioisotopic method (Furukawa et al. 1987, Vadas et al. 1975). Briefly, mice were sensitized to 2,4-dinitrofluorobenzene (DNFB; Sigma) by applying 50 µL of 2% DNFB solution in ethanol to their shaved backs. After 5 d, 10 µL of 1% DNFB solution in olive oil was applied on both sides of the right ear and olive oil on the left ear as a control. Ten hours after the second administration of DNFB, mice were injected intraperitoneally with 74 µBq [¹²⁵I]-indo-2-deoxyuridine (Amersham, Arlington Heights, IL). Sixteen hours later, mice were killed and both ears were cut off at the hairline and counted in a Cobra II auto-gamma counter (Packard, Meriden, CT). The results were calculated as follows:

Antibody response to sheep red blood cells (SRBC).  Antibody response to SRBC was measured by the method of Herbert (1978). Briefly, sheep blood was collected with heparinized tubes and centrifuged at 800 × g for 10 min. After the serum and buffy coat were removed, SRBC were washed three times with sterile PBS. SRBC were suspended at 2.5 × 10¹² cells/L in PBS and 0.2 mL was injected into mice intraperitoneally. Five days after the challenge, mice were killed and the serum was collected for measuring antibody titer by a hemagglutination assay (Herbert 1978).

Statistical analysis.  Data were analyzed for overall age and treatment effects by two-way ANOVA. Individual mean differences were determined by Tukey's Honestly Significant Difference test (SYSTAT 1992). All statistical analyses were conducted using SYSTAT for Macintosh version 5.2 (SYSTAT, Evanston, IL). Data are reported as means ± SEM. Differences at P < 0.05 were considered significant.

	RESULTS

Abstract Introduction Methods Results Discussion References

One mouse fed 1.0% PSP died before the completion of dietary intervention. Four mice (one fed control diet, two fed 0.5% PSP and one fed 1.0% PSP) were excluded from the study because of the presence of visible tumors or splenomegaly. Body weights were not significantly affected by dietary PSP supplementation (data not shown). There was no difference in histopathologic abnormalities between mice supplemented with PSP or those fed control diet (data not shown).

Mitogenic response.  The optimal concentrations for stimulating splenocyte proliferation were 1.5, 5 and 15 mg/L for Con A, PHA and LPS, respectively. Young and old mice had the same optimal concentrations for all mitogens used, an outcome that was not affected by the diets. Table 2 shows proliferation in response to optimal concentrations of mitogens. As previously reported (Miller 1991), old mice had significantly lower lymphocyte proliferation in response to T cell mitogens Con A and PHA compared with young mice. When cells were stimulated with optimal or supraoptimal concentrations of T cell mitogens, PSP did not have a significant effect on splenocyte proliferation (Table 2 and data not shown). However, when cells were stimulated by a suboptimal concentration of Con A (0.5 mg/L), young mice fed 1.0% PSP had significantly greater splenocyte proliferation compared with those fed the control diet (79,909 ± 17,270 vs. 43,798 ± 13,998 ccpm, P < 0.05). When splenocytes were stimulated by B-cell mitogen LPS, no significant changes were observed as a result of PSP supplementation or age (Table 2). Similar results were also observed with the use of suboptimal or supraoptimal concentrations of LPS (data not shown).

Cytokine and PGE₂ production.  No age-related difference in LPS-stimulated IL-1 production was observed. Supplementation with PSP at any tested levels did not affect IL-1 production (data not shown). PHA-stimulated IL-2 production was significantly lower in the splenocytes from old mice than in those from young mice (51.2 ± 9.9 vs. 150.8 ± 18.1 ng/L, P < 0.001). PSP did not have a significant effect on IL-2 production (data not shown). The Con A- stimulated splenocytes from old mice produced significantly more IL-4 than those from young mice (24.2 ± 4.6 vs. 14.3 ± 1.6 ng/L, P < 0.05). PSP did not significantly change IL-4 levels in either young or old mice (data not shown). The splenocytes from old mice synthesized significantly higher levels of PGE₂ compared with those from young mice (1338 ± 241 vs. 807 ± 122 ng/L, P < 0.05). PSP had no significant effect on PGE₂ production at any supplementation level (data not shown).

Delayed-type hypersensitivity.  As demonstrated in Figure 1, there was no significant difference in DTH between young and old mice. The age/diet interaction was significant, and post-hoc tests showed that old mice fed 1.0% PSP had significantly higher DTH response than those fed the control diet, whereas no such effect was observed in young mice.

View larger version (27K): [in this window] [in a new window]   Fig 1. Effect of protein-bound polysaccharides (PSP) supplementation (1%) on delayed-type hypersensitivity (DTH) skin response of mice. Mice were sensitized and challenged with 2,4-dinitrofluorobenzene (DNFB) and injected with [125I]-deoxyuridine as described in Materials and Methods. Stimulation index was defined as the ratio of cpm of the stimulated ear minus background to cpm of the control ear minus background. Each bar represents mean ± SEM of 10 mice. Two-way ANOVA showed a significant age/diet interaction. *Significantly greater than old mice fed control diet, P < 0.05 (Tukey's Honestly Significant Difference test).

Antibody response to SRBC.  Overall, young mice produced higher levels of antibody against SRBC than old mice (1.84 ± 0.06 vs. 1.44 ± 0.18 log antibody titer, P < 0.05). PSP supplementation did not significantly affect this response in either young or old mice.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

BRM have been shown to potentiate immune function and increase resistance to infections and cancers (de Ojeda et al. 1994, Matsunaga et al. 1992b, Peterson et al. 1994). Protein-bound polysaccharides originating from some edible fungi such as mushrooms and with the common active component -[1-3]-glucan are considered effective BRM (Gomma et al. 1992, Jong and Birmingham 1993, Matsunaga et al. 1992b, Yang et al. 1993). Most studies of these polysaccharides have been conducted with a focus on cancer therapy. Polysaccharides such as krestin (PSK) or lentinan have been shown to have inhibitory effects on tumor growth and to extend survival time of tumor-bearing animals and cancer patients (Gomma et al. 1992, Hamuro et al. 1994, Matsunaga et al. 1992b, Suzuki et al. 1994). PSP used in this study is similar to PSK (both extracted from the mycelia of Coriolus versicolor but different strains) and has also been shown to have systemic immunoenhancing and antineoplastic properties (Wang et al. 1993, Xu et al. 1993).

In this study, except for a significant increase in mitogenic response to suboptimal concentration of Con A as a result of 1.0% dietary PSP supplementation in young mice, there were no significant effects of PSP (0.1-1.0%) on lymphocyte proliferation stimulated by T and B cell mitogens, or cytokine (IL-1, IL-2, IL-4) and PGE₂ production. Because the improvement in young mice was observed with only one dose of mitogen and in only one in vitro immune response test, we do not believe that this finding indicates an immunoenhancing effect in young mice. This agrees with the results reported by Hashimoto et al. (1991) in which oral administration of -[1-3]-glucan did not affect mitogenic response of spleen cells in mice. In contrast, others using polysaccharides of different origins have reported enhancement of lymphocyte proliferation (Gomma et al. 1992, Hirai et al. 1993, Sugimachi et al. 1995, Suzuki et al. 1989). The discrepancy could be due to the difference in design and number of animals used, the source of the polysaccharides, the route of administration as well as the species and health status of animals used in the different experiments. It should be pointed out that in most studies in which an enhancement of the immune response is reported, tumor-bearing animals or subjects with cancer or infection were used (Gomma et al. 1992, Kaneko and Chihara 1992, Noguchi et al. 1995).

No significant effect was observed on antibody response to SRBC used as an in vivo measure of T cell-dependent humoral immune response. In young mice, no significant effect of PSP was observed on DTH, but old mice fed 1.0% PSP had a significantly higher DTH compared with those fed the control diet. Suzuki et al. (1994) reported that administration of lentinan to tumor-inoculated BDF₁ mice significantly enhanced DTH response against tumor-associated antigens. In another study conducted by Matsunaga et al. (1992b), intraperitoneal administration of PSK at 10-500 mg/kg did not affect DTH response of young healthy C57BL/6 mice, whereas PSK at 250 mg/kg prevented the decrease in DTH response caused by the tumor burden in tumor-bearing mice. Thus polysaccharides appear to improve DTH in immunocompromised hosts such as aged or tumor-bearing animals. In both cases, overproduction of immunosuppressive factors was observed (Matsunaga et al. 1992a and 1992b, Hayek et al. 1994). It has been proposed that PSP, at least in the case of tumor-bearing animals, reduces production of immunosuppressive factors (Matsunaga et al. 1992a and 1992b). In this study, however, although old mice were shown to have higher PGE₂ production, PSP had no effect on production of this immunosuppressive factor. It is possible, however, that PSP can reduce formation of other suppressive factors not measured in this study. Contrary to the increased DTH response in old mice by PSP supplementation, PSP had no effect on lymphocyte proliferation, an in vitro index of T cell-mediated function. This might be due to use of fetal bovine serum in cultures rather than autologous serum, one or more of whose components could be affected by PSP in vivo. On the other hand, PSP could affect other immunoregulatory mechanisms involved in DTH not represented by in vitro indices measured here. The biological significance of the 25% increase in DTH of old mice observed in this study has to be determined, because data to construct a correlative relationship between various degrees of change in DTH and health outcome are not available. However, studies have shown that anergy or relative anergy in DTH is associated with increased morbidity and mortality (Christou et al. 1989, Wayne et al. 1990). The mechanism by which PSP-containing mushroom extract increases DTH was not determined. Two possibilities exist. One is that the simple molecules of carbohydrates (monosaccharides and disaccharides) resulting from digestion of the complex polysaccharides exert the biological effects. However, there are no reports related to immunologic effects of simple carbohydrates on immune response. The other possibility is that the complex carbohydrates exert an effect on gut-associated immunity before absorption, which is then transferred to the systemic immune response via lymph node and peripheral blood. The lack of more pronounced effects in this study was not due to use of inadequate levels of these compounds because the wide range of levels (0-1 g/100 g diet) used contained and exceeded doses recommended by manufacturers for human use and those reported by other investigators to exert immunoenhancing and antitumor effects (see Materials and Methods section).

In summery, our results indicate that oral supplementation with PSP had a modest immunoenhancing effect on in vivo T cell-mediated function in old mice, whereas no effect was observed on in vitro T cell-mediated function or in vivo B cell-mediated function in young or old mice. Thus polysaccharide supplementation might be beneficial in modestly enhancing the immune response in old mice with suppressed T cell-mediated functions, but it does not seem to have an effect on the immune response of healthy young mice. These data in young and old mice do not support the claims of general systemic immunoenhancing effects for PSP-containing mushroom extracts consumed orally. It is possible, however, that these compounds might have an independent effect on gut-associated immunity.

	ACKNOWLEDGMENTS

The authors thank Alison Beharka and Michelle S. Santos for their review of the manuscript and Timothy S. McElreavy for preparation of this manuscript.

	FOOTNOTES

Manuscript received 28 April 1997. Initial reviews completed 2 July 1997. Revision accepted 4 September 1997.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• American Institute of Nutrition Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977; 107:1340-1348
• Bogden J. D., Bendich A., Kemp F. W., Bruening K. S., Skurnick J. H., Denny T., Baker H., Louria D. B. Daily micronutrient supplements enhance delayed-hypersensitivity skin test responses in older people. Am. J. Clin. Nutr. 1994; 60:437-447 [Abstract/Free Full Text]
• Chandra R. K. Effect of vitamin and trace-element supplementation on immune responses and infections in elderly subjects. Lancet 1992; 340:1124-1127 [Medline]
• Christou N. V., Tellado-Rodriguez J., Chartrand L., Giannas B., Kapadia B., Meakins J., Rode H., Gordon J. Estimating mortality risk in preoperative patients using immunologic, nutritional, and acute-phase response variables. Ann. Surg. 1989; 210:69-77 [Medline]
• de Ojeda G., Diez-Orejas R., Portoles P., Ronda M., Del Pozo M. L., Feito M. J., Hartleb M., Rojo J. M. Polyerga, a biological response modifier enhancing T-lymphocyte-dependent responses. Res. Exp. Med. 1994; 194:261-271 [Medline]
• Furukawa T., Meydani S. N., Blumberg J. B. Reversal of age-associated decline in immune responsiveness by dietary glutathione supplementation in mice. Mech. Aging Dev. 1987; 38:107-117
• Gomma K., Kraus J., Robkopf H., Roper H., Franz G. Antitumour and immunological activity of a 1-3/1-6 glucan from Glomerella cingulata. J. Cancer Res. Clin. Oncol. 1992; 118:136-140 ^[Medline]
• Hamuro J., Takatsuki F., Suga T., Kikuchi T., Suzuki M. Synergistic antimetastatic effects of lentinan and interleukin 2 with pre- and post-operative treatments. Jpn. J. Cancer Res. 1994; 85:1288-1297 [Medline]
• Hashimoto K., Suzuki I., Yadomae T. Oral administration of SSG, a beta-glucan obtained from Sclerotinia sclerotiorum, affects the function of Peyer's patch cells. Int. J. Immunopharmacol. 1991; 13:437-442 [Medline]
• Hayek M. G., Meydani S. N., Meydani M., Blumberg J. B. Age differences in eicosanoid production of mouse splenocytes: effects on mitogen-induced T-cell proliferation. J. Gerontol. 1994; 49:B197-B207 [Abstract]
• Herbert, W. J. (1978) Passive hemagglutination with special reference to the tanned cell technique. In: Handbook of Experimental Immunology (Weir, D. M., ed.), pp. 20.1-20.20. Blackwell Scientific Publications, Oxford, UK.
• Hirai R., Oguchi Y., Sugita N., Matsunaga K., Nomoto K. Enhancement of T-cell proliferation by PSK. Int. J. Immunopharmacol. 1993; 15:745-750 [Medline]
• Jong S. C., Birmingham J. M. Medicinal and therapeutic value of the Shiitake mushroom. Adv. Appl. Microbiol. 1993; 39:153-184 [Medline]
• Kaneko, Y. & Chihara, G. (1992) Potentiation of host resistance against microbial infections by lentinan and its related polysaccharides. In: Microbial Infections. (Friedman, H.E.A., ed.), pp. 201-215. Plenum Press, New York, NY.
• Matsunaga K., Iijima H., Aota M., Oguchi Y., Fujii T., Yoshikumi C., Nomoto K. Enhancement of effector cell activities in mice bearing syngeneic plasmacytoma X5563 by biological response modifier, PSK. J. Clin. Lab. Immunol. 1992; 37:21-37 [Medline]
• Matsunaga K., Morita I., Iijima H., Oguchi Y., Yoshimura M., Fujii T., Yoshikumi G., Nomoto K. Effects of biological response modifiers with different modes of action used separately and together on immune response in mice with syngeneic tumours. J. Int. Med. Res. 1992; 20:406-421 [Medline]
• Matsunaga K., Morita I., Oguchi Y. Restoration of immune responsiveness by a biological response modifier, PSK, in aged mice bearing syngeneic transplantable tumor. J. Clin. Lab. Immunol. 1987; 24:143-149 [Medline]
• McCosh E. J., Meyer D. L., Dupont J. Radioimmunoassay of prostaglandin E₁, E₂, F₂ in unextracted plasma, serum and myocardium. Prostaglandins 1976; 12:476-486
• Meydani S. N., Barklund M. P., Liu S., Meydani M., Miller R. A., Cannon J. G., Moorow F. D., Docklin R., Blumberg J. B. Vitamin E supplementation enhances cell-mediated immunity in healthy elderly subjects. Am. J. Clin. Nutr. 1990; 52:557-563 [Abstract/Free Full Text]
• Meydani S. N., Dupont J. Effect of zinc deficiency on prostaglandin synthesis in different organs of the rat. J. Nutr. 1982; 112:1098-1104
• Meydani S. N., Shapiro A. C., Meydani M., Macauley J. B., Blumberg J. B. Effect of age and dietary fat (fish, corn and coconut oils) on tocopherol status of C57BL/6NIA mice. Lipids 1987; 22:345-350 [Medline]
• Miller R. A. Aging and immune function. Int. Rev. Cytol. 1991; 124:187-215 [Medline]
• Noguchi K., Tanimura H., Yamaue H., Tsunoda T., Iwahashi M., Tani M., Mizobata S., Hotta T., Arii K., Tamai M. Polysaccharide preparation PSK augments the proliferation and cytotoxicity of tumor-infiltrating lymphocytes in vitro. Anticancer Res. 1995; 15:255-258 [Medline]
• Penna C., Dean P. A., Nelson H. Pulmonary metastases neutralization and tumor rejection by in vivo administration of  glucan and bispecific antibody. Int. J. Cancer 1996; 65:377-382 [Medline]
• Peterson V. M., Adamovicz J. J., Elliott T. B., Moore M. M., Madonna G. S., Jackson W.E.R., Ledney G. D., Gause W. C. Gene expression of hematoregulatory cytokine is elevated endogenously after sublethal gamma irradiation and is differentially enhanced by therapeutic administration of biologic response modifiers. J. Immunol. 1994; 153:2321-2330 [Abstract]
• Sugimachi K., Maehara Y., Kusumoto T., Okamura T., Korenaga D., Kohnoe S., Baba H., Anai H. In vitro reactivity to a protein-bound polysaccharide PSK of peripheral blood lymphocytes from patients with gastrointestinal cancer. Anticancer Res. 1995; 15:2175-2179 [Medline]
• Suzuki I., Hashimoto K., Ohno N., Tanaka H., Yadomae T. Immunomodulation by orally administered -glucan in mice. Int. J. Immunopharmacol. 1989; 11:761-769 [Medline]
• Suzuki M., Iwashiro M., Takatsuki F., Kuribayashi K., Hamuro J. Reconstitution of anti-tumor effects of Lentinan in nude mice: role of delayed-type hypersensitivity reaction triggered by CD4-positive T cell clone in the infiltration of effector cells into tumor. Jpn. J. Cancer Res. 1994; 85:409-417 [Medline]
• SYSTAT (1992) Statistics, Version 5.2. SYSTAT, Evanston, IL.
• Vadas M. A., Miller J.F.A.P., Gamble J., Whitelaw A. A radioisotopic method to measure delayed-type hypersensitivity in the mouse. Int. Arch. Allergy Appl. Immunol. 1975; 49:670-692 [Medline]
• Wang, C.-A., Ma, A.-L. & Gao, Y. (1993). The Effect of Yun Zhi Essence on Tumor Inhibition and Anti-Metastasis of Mice. PSP International Symposium 1993, Hong Kong, Fudan University Press.
• Wayne S. J., Rhyne R. L., Garry P. J., Goodwin J. S. Cell-mediated immunity as a predictor of morbidity and mortality in the aged. J. Gerontol. Med. Sci. 1990; 45:M45-M48
• Xu, M.-F., Gu, Z.-L., Qhian, Z.-N., Chou, W.-H. & Kwok, C.-Y. (1993). The Effect of PSP on the Immunological Function of Rats. PSP International Symposium 1993, Hong Kong, Fudan University Press.
• Yang, Q.-Y., Hu, Y.-J., Li, X.-Y., Yang, J.-C., Liu, J.-X., Liu, T.-F., Xu, G.-M. & Liao, M.-L. (1993). A New Biological Response Modifier SubstancePSP. PSP International Symposium 1993, Hong Kong, Fudan University Press.

0022-3166/98 $3.00 ©1998 American Society for Nutritional Sciences