Enhanced therapeutic anti-tumor immunity induced by co-administration of 5-fluorouracil and adenovirus expressing CD40 ligand

Bystander immune activation by chemotherapy has recently gained extensive interest and provided support for the clinical use of chemotherapeutic agents in combination with immune enhancers. The CD40 ligand (CD40L; CD154) is a potent regulator of the anti-tumor immune response and recombinant adenovirus (RAd)-mediated CD40L gene therapy has been effective in various cancer models and in man. In this study we have assessed the combined effect of local RAd-CD40L and 5-fluorouracil (5-FU) administration on a syngeneic MB49 mouse bladder tumor model. Whereas MB49 cells implanted into immunocompetent mice responded poorly to RAd-CD40L or 5-FU alone, administration of both agents dramatically decreased tumor growth, increased survival of the mice and induced systemic MB49-specific immunity. This combination treatment was ineffective in athymic nude mice, highlighting an important role for T cell mediated anti-tumor immunity for full efficacy. 5-FU up-regulated the expression of Fas and immunogenic cell death markers in MB49 cells and cytotoxic T lymphocytes from mice receiving RAd-CD40L immunotherapy efficiently lysed 5-FU treated MB49 cells in a Fas ligand-dependent manner. Furthermore, local RAd-CD40L and 5-FU administration induced a shift of myeloid-derived suppressor cell phenotype into a less suppressive population. Collectively, these data suggest that RAd-CD40L gene therapy is a promising adjuvant treatment to 5-FU for the management of bladder cancer.     

Immune Depletion in Combination with Allogeneic Islets Permanently Restores Tolerance to Self-Antigens in Diabetic NOD Mice

The destruction of beta cells in type 1 diabetes (T1D) results in loss of insulin production and glucose homeostasis. Treatment of non-obese diabetic (NOD) mice with immune-depleting/modulating agents (e.g., anti-CD3, murine anti-thymocyte-globulin (mATG)) can lead to diabetes reversal. However, for preclinical studies with these and other agents seeking to reverse disease at onset, the necessity for exogenous insulin administration is debated. Spontaneously diabetic NOD mice were treated with a short-course of mATG and insulin provided as drug therapy or by way of allogeneic islet implants. Herein we demonstrate that exogenous insulin administration is required to achieve disease reversal with mATG in NOD mice. Unexpectedly, we also observed that provision of insulin by way of allogeneic islet implantation in combination with mATG leads to a pronounced reversal of diabetes as well as restoration of tolerance to self-islets. Expansion/induction of regulatory cells was observed in NOD mice stably cured with mATG and allogeneic islets. These data suggest that transient provision of allogeneic insulin-producing islets might provide a temporary window for immune depletion to be more effective and instilling stable tolerance to endogenous beta cells. These findings support the use of a never before explored approach for preserving beta cell function in patients with recent onset T1D.     

The release of DNA into the plasma of mice following hepatic cell death by apoptosis and necrosis

The goal of these investigations was to measure levels of DNA in the plasma of mice following administration of hepatotoxic agents to induce apoptotic or necrotic cell death and determine any differences in the release of this marker depending upon death pathway. For this purpose, the effects of varying doses of anti-Fas, acetaminophen (APAP) or carbon tetrachloride (CCl₄) were assessed in normal mice. Plasma DNA was measured fluorometrically by the dye PicoGreen while lactate dehydrogenase (LDH) and caspase 3, other molecules released with cell injury or death, were measured by enzymatic assays. Histology was used to assess the occurrence of apoptosis or necrosis. Results of these experiments indicate that increased blood DNA levels occurred with all three agents and were highest with anti-Fas and CCl₄; caspase 3 levels were much higher with anti-Fas than the other agents. Histological examination confirmed the predominance of apoptotic death with anti-Fas and necrotic death with APAP and CCl₄. These results indicate that increased blood DNA is common in hepatotoxic injury and is a feature of both apoptotic and necrotic death.     

Effects of morphine on the phenotypic expression of cell surface markers on murine lymphocytes.

There is a growing literature indicating that opioid abuse by human addicts and opioid administration to animals have profound effects on the immune system. In the present study, implantation of morphine pellets in mice was associated with reduced phenotypic expression of the cell surface antigens specific to T-lymphocytes and to helper and cytotoxic/suppressor T-lymphocyte subtypes. The effect of morphine, as measured by flow cytometry using monoclonal antibodies specific for antigens expressed by these cells, was dose-dependent. The decrease in expression of antigens was apparent as early as 24 h after morphine pellet implantation and continued for 3 days. In addition, a time-dependent increase in the expression of these antigens was observed in placebo- and morphine-treated mice, suggesting that the pellets had a small antigenic effect. However, at all times studied, morphine-treated mice had fewer cells expressing the antigens than placebo-treated mice. Our results provide additional evidence that the use of opioids by IV drug abusers compromises their immune function.     

The release of DNA into the plasma of mice following hepatic cell death by apoptosis and necrosis

The goal of these investigations was to measure levels of DNA in the plasma of mice following administration of hepatotoxic agents to induce apoptotic or necrotic cell death and determine any differences in the release of this marker depending upon death pathway. For this purpose, the effects of varying doses of anti-Fas, acetaminophen (APAP) or carbon tetrachloride (CCl₄) were assessed in normal mice. Plasma DNA was measured fluorometrically by the dye PicoGreen while lactate dehydrogenase (LDH) and caspase 3, other molecules released with cell injury or death, were measured by enzymatic assays. Histology was used to assess the occurrence of apoptosis or necrosis. Results of these experiments indicate that increased blood DNA levels occurred with all three agents and were highest with anti-Fas and CCl₄; caspase 3 levels were much higher with anti-Fas than the other agents. Histological examination confirmed the predominance of apoptotic death with anti-Fas and necrotic death with APAP and CCl₄. These results indicate that increased blood DNA is common in hepatotoxic injury and is a feature of both apoptotic and necrotic death.     

Nonspecific stimulation of the maternal immune system. I. Effects On teratogen-induced fetal malformations

BACKGROUND: Maternal immune stimulation has reported, but unconfirmed, efficacy for reducing chemical-induced morphologic defects in mice. METHODS: Teratogenic chemicals (2,3,7, 8-tetrachlorodibenzo-p-dioxin [TCDD], ethyl carbamate [urethane], methylnitrosourea [MNU], or valproic acid [VA]) were given to pregnant mice to induce cleft palate (TCDD, urethane), digital defects (urethane, MNU), or exencephaly (VA). Before teratogen administration, the immune system of female mice was stimulated by intraperitoneal (IP) administration of pyran copolymer or attenuated bacillus Calmette Guérin (BCG), or by footpad injection with Freund's complete adjuvant (FCA). RESULTS: Fetal defects caused by all four chemicals studied were reduced by maternal immunostimulation, sometimes dramatically. In addition to reducing VA-induced exencephaly, immunostimulation with FCA resulted in fetal mice displaying anury (absence of tails). Activated maternal immune cells could not be detected in fetal circulation using flow cytometry and a fluorescent cell-tracking probe. CONCLUSIONS: For the chemicals tested, maternal immune stimulation has efficacy in reducing fetal defects. Immune protection against teratogenesis may be an indirect effect of maternal immune cell activation.     

Immunoenhancing effects of alprazolam in mice

The GABA/benzodiazepine receptor chloride channel complex has been proposed to play a modulatory role in immune function. The effects of alprazolam, a triazolobenzodiazepine with high affinity for "central" benzodiazepine receptors, were examined on several parameters of immune function in mice. Natural killer cell activity, mixed leukocyte reactivity and mitogen-induced lymphocyte proliferation were all significantly increased two hr after administration of low doses (0.02-1.0 mg/kg) of alprazolam. Twenty four hr later, similar but less robust immunoenhancing effects were observed. Higher doses of alprazolam (5-10 mg/kg) did not affect these measures of immune function. The immunoenhancing effects of alprazolam did not appear related to corticosterone levels. In contrast, vehicle injection caused a profound suppression of these immune parameters two hr later, which was no longer apparent 24 hr later. This immunosuppression appeared to correlate with a concurrent rise in serum corticosterone levels. These data support the hypothesis that the GABA/benzodiazepine receptor chloride channel complex modulates immune function. Use of low doses of alprazolam may be of potential clinical importance when immunoenhancement is required.     

Inhibition of spontaneous pulmonary metastases of Lewis lung carcinoma by oral treatment with Respivax and Broncho-Vaxom

Summary The antimetastatic activity of orally administered polybacterial vaccines, Broncho-Vaxom (BV) and Respivax (RV) was examined in C57BL/6 mice, bearing implants of Lewis lung carcinoma (3LL) in the footpad. The oral administration of BV or RV for 10 consecutive days before or after surgery caused significant reduction of the number and volume of lung metastases. In addition, the therapeutic potential of BV and RV was examined in combination with chemotherapy to determine if there is additive activity. In animals bearing pulmonary micrometastases, treatment with a combination of cyclophosphamide at 50–150 mg/kg with BV or RV was found to be more effective than each of these treatments alone. In immune function studies it was established that the oral administration of BV and RV induced an increase in the number of cells, recovered by broncho-alveolar lavage, and alveolar macrophages were dominant in these cell populations. Furthermore, oral treatment of mice with these vaccines rendered their alveolar macrophages tumoricidal for syngeneic metastatic 3LL cells in vitro. These results show that pulmonary macrophages induced by oral administration of BV and RV played a key role in the inhibition of metastasis in 3LL-bearing mice.     

Prevention of immunosuppression in stressed mice by neurotropin(NSP)

It is well known that the immune function can be compromised by stress. To investigate immune function in mice stressed by experimental restraint or unavoidable and opioid dependent stress, we evaluated the changes in total body weight and in organ weights (liver, spleen and thymus) of these animals, as well as the phagocytic activity of macrophages, the cytotoxicity of T cells and inhibitory effects on tumor growth and changes in T cell subset populations. At the same time we evaluated the effects of Neurotropin (NSP), a substance extracted from the inflamed skin of rabbits inoculated with the vaccinia virus and which appears to possess neuroimmunomodulating activity. The experimentally stressed group exhibited a reduction of phagocytic activity of macrophages, cytotoxicity of T cells and inhibitory effects on tumor growth. In addition there were changes in the population of T cell subsets. In those animals pretreated with NSP, the immunosuppression induced by stress was ameliorated. As compared with several agents which influence phagocytosis, neurotropin exhibited effects similar to that of agents that blocked the adrenaline receptor and an opioid antagonist rather than tranquilizer (diazepam) and a cholinergic receptor blocker. The pharmacologic effects of neurotropin support a relationship between the actions of the central nervous system and the immune system.     

Effects of single or repeated administrations of methamphetamine on immune response in mice.

The present study aimed to clarify the connection between immune responses and the administration frequency of methamphetamine (MAP) in male and female mice. Male and female ddY mice were given single or multiple (repeated for 10 days) intraperitoneal injections of MAP (5.0 mg/kg/day). The following immune parameters were examined; the number of leukocytes in peripheral blood and the proliferative activity (phytohemagglutinin;PHA, lipopolysaccharide; LPS response) and natural killer (NK) cell activity in splenic lymphocytes. Further, the differences in metabolic function in the spleen in response to MAP (and its metabolite amphetamine) in male and female mice were measured by gas chromatography. The results of the present study were that; 1) single and repeated MAP injections reduced leukocytes; 2) single MAP injection increased the proliferative response of splenic lymphocytes to PHA stimulation in only male mice, but the response to LPS stimulation was slightly increased in both male and female mice; 3) single and repeated MAP injections reduced NK cell activity of splenic lymphocytes, and especially in female mice with 5 injections of MAP; 4) with 10 MAP injections the NK cell activity and leukocytes recovered to the level of controls; and 5) the metabolic activity of MAP was reduced in female mice treated acutely with MAP in comparison to male mice. These results appear to indicate that immune responses to MAP were involved in the different results shown for administration frequency, sex difference and metabolic process of MAP.     

MUC1-specific immune responses in human MUC1 transgenic mice immunized with various human MUC1 vaccines

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen. Received: 7 July 1999 / Accepted: 26 August 1999     

Melatonin enhancement of splenocyte proliferation is attenuated by luzindole, a melatonin receptor antagonist

In addition to marked seasonal changes in reproductive, metabolic, and other physiological functions, many vertebrate species undergo seasonal changes in immune function. Despite growing evidence that photoperiod mediates seasonal changes in immune function, little is known regarding the neuroendocrine mechanisms underlying these changes. Increased immunity in short days is hypothesized to be due to the increase in the duration of nightly melatonin secretion, and recent studies indicate that melatonin acts directly on immune cells to enhance immune parameters. The present study examined the contribution of melatonin receptors in mediating the enhancement of splenocyte proliferation in response to the T cell mitogen Concanavalin A in mice. The administration of luzindole, a high-affinity melatonin receptor antagonist, either in vitro or in vivo significantly attenuated the ability of in vitro melatonin to enhance splenic lymphocyte proliferation during the day or night. In the absence of melatonin or luzindole, splenocyte proliferation was intrinsically higher during the night than during the day. In the absence of melatonin administration, luzindole reduced the ability of spleen cells to proliferate during the night, when endogenous melatonin concentrations are naturally high. This effect was not observed during the day, when melatonin concentrations are low. Taken together, these results suggest that melatonin enhancement of splenocyte proliferation is mediated directly by melatonin receptors on splenocytes and that there is diurnal variation in splenocyte proliferation in mice that is also mediated by splenic melatonin receptors.     

NK and CD4 cells collaborate to protect against melanoma tumor formation in the brain

NK cells represent a potent immune effector cell type that have the ability to recognize and lyse tumors. However, the existence and function of NK cells in the traditionally "immune-privileged" CNS is controversial. Furthermore, the cellular interactions involved in NK cell anti-CNS tumor immunity are even less well understood. We administered non-Ag-loaded, immature dendritic cells (DC) to CD8alpha knockout (KO) mice and studied their anti-CNS tumor immune responses. DC administration induced dramatic antitumor immune protection in CD8alpha KO mice that were challenged with B16 melanoma both s.c. and in the brain. The CNS antitumor immunity was dependent on both CD4+ T cells and NK cells. Administration of non-Ag-loaded, immature DC resulted in significant CD4+ T cell and NK cell expansion in the draining lymph nodes at 6 days postvaccination, which persisted for 2 wk. Finally, DC administration in CD8alpha KO mice was associated with robust infiltration of CD4+ T cells and NK cells into the brain tumor parenchyma. These results represent the first demonstration of a potent innate antitumor immune response against CNS tumors in the absence of toxicity. Thus, non-Ag-loaded, immature DC administration, in the setting of CD8 genetically deficient mice, can induce dramatic antitumor immune responses within the CNS that surpass the effects observed in wild-type mice. Our results suggest that a better understanding of the cross-talk between DC and innate immune cells may provide improved methods to vaccinate patients with tumors located both systemically and within the CNS.     

Manganese superoxide dismutase expressed in silkworm larvae,Bombyx mori L enhances the NK activity and splenocyte proliferation against Sarcoma 180 tumor cells in vivo

Natural killer cell (NK) is known as a major immune system in body through mediating cell death via several possible pathways, and one of three subpopulations of lymphocytes functioning as scavenger of tumor, virus infected cells etc. Our present results found that the SOD-contained silkworm larvae powder caused an enhancement of the effect on NK cell cytotoxicity, which implied this material modulated the immune system in mice in vivo. The NK cell activities of S180 tumor modeled mice treated with silkworm powder including SOD were enhanced significantly ranging from 30% to 48%, respectively, compare to a distilled water feeding control and silkworm powder without SOD. Meanwhile, the ConA-stimulated splenocyte proliferation of all three treated groups was higher than that of the control both in T cells or B cells. The average tumor weight of S180 modeled mice treated with doses of SOD-contained silkworm powder was lighter than that of water control showing the tumor inhibition rates (IR) reached to 22.51% to 37%, respectively. In conclusion, these findings demonstrate that administration of silkworm larvae powder containing SOD results in activation of NK cells and immune T-cell and B-cell, suggesting the silkworm larvae powder containing SOD play a positive role in tumor inhibition.     

Natural killer cells and mast cells from gp49B null mutant mice are functional

Immune responses are controlled by a combination of positive and negative cellular signals. Effector cells in the immune system express inhibitory receptors that serve to limit effector cell expansion and to protect the host from autoreactivity. gp49B is a receptor of unknown function that is expressed on activated mast cells and natural killer (NK) cells and whose cytoplasmic tail endows it with inhibitory potential. To gain insight into the function of gp49B in mice, we disrupted the gp49B gene by homologous recombination. gp49B(0) mice were born at expected ratios, were healthy and fertile, and displayed normal long-term survival rates. gp49B(0) mice showed no defect in NK or mast cell development. Furthermore, NK and mast cells from the gp49B(0) mice showed activation properties in vitro similar to those of cells isolated from wild-type mice. Therefore, gp49B is not critical for the development, expansion, and maturation of mast cells and NK cells in vivo. The healthy status of gp49B(0) mice makes them suitable for testing the role of gp49B in immune responses to infectious agents.     

Immunoregulative effects of carnosine and beta-alanine

Physiological factors involved in immunity and tissue repair with regulate homeostasis, a physiological function of the connective tissue, are as yet unidentified. We earlier detected the granulation-promoting action of carnosine, and reported on the acceleration of tissue repair in experimental as well as clinical studies. In that study, immunoregulatory effects of carnosine and beta-alanine were examined by the plaque-forming cell (PFC) count and delayed hypersensitivity reaction (DHR). The PFC value increased in mice pretreated with these agents. In these mice, PFC reaction to 2 X 10(7) SRBC was enhanced but that to 1 X 10(9) SRBC was suppressed. The agents also suppressed excess immunoreaction in immature mice but increased weakened immunoreaction in aged animals. Furthermore, the agents had the optimal doses for the enhancement of both PFC reaction to 1 X 10(8) SRBC and DHR to 1% picryl chloride. They also induced recovery of immunofunction suppressed by the administration of MMC. Carnosine and beta-alanine exerts immunoregulatory effects by activating both T and B cells. Our observations indicated that the agents not only promote tissue repair but also help maintain homeostasis and accelerate spontaneous healing.     

Chronobiology in hematology and immunology

The hematopoietic and the immune systems in all their components are characterized by a multifrequency time structure with prominent rhythms in cell proliferation and cell function in the circadian, infradian, and rhythms in cell proliferation and cell function in the circadian, infradian, and circannual frequency ranges. The circulating formed elements in the peripheral blood show highly reproducible circadian rhythms. The timing and the extent of these rhythms were established in a clinically healthy human population and are shown as chronograms, cosinor summaries and, for some high-amplitude rhythms, as time-qualified reference ranges (chronodesms). Not only the number but also the reactivity of circulating blood cells varies predictably as a function of time as shown for the circadian rhythm in responsiveness of human and murine lymphocytes in vitro to lectin mitogens (phytohemagglutinin and pokeweed mitogen). Some circadian rhythms of hematologic functions appear to be innate and are presumably genetically determined but are modulated and adjusted in their timing by environmental factors, so-called synchronizers. Phase alterations in the circadian rhythms of hematologic parameters of human subjects and of mice by manipulation of the activity-rest or light-dark schedule and/or of the time of food uptake are presented. Characteristically these functions do not change their timing immediately after a shift in synchronizer phase but adapt over several and in some instances over many transient cycles. The circadian rhythm of cell proliferation in the mammalian bone marrow and lymphoid system as shown in mice in vivo and in vitro may lend itself to timed treatment with cell-cycle-specific and nonspecific agents in an attempt to maximize the desired and to minimize the undesired treatment effects upon the marrow. Differences in response, and susceptibility of cells and tissues at different stages of their circadian and circaseptan (about 7-day) rhythms and presumably of cyclic variations in other frequencies are expected to lead to the development of a chronopharmacology of the hematopoietic and immune system. Infradian rhythms of several frequencies have been described for numerous hematologic and immune functions. Some of these, i.e., in the circaseptan frequency range, seem to be of importance for humoral and for cell mediated immune functions including allograft rejection. Infradian rhythms with periods of 19 to 22 days seem to occur in some hematologic functions and are very prominent in cyclic neutropenia and (with shorter periods) in its animal model, the grey collie syndrome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synergism for cytokine-mediated disease during concurrent endotoxin and viral challenges: roles for NK and T cell IFN-gamma production

Viral infections in humans or mice can result in increased sensitivity to challenges with bacteria, bacterial products, or cytokine administration. During lymphocytic choriomeningitis virus infections, mice are more sensitive to the lethal effects of bacterial endotoxin LPS, and in the experiments reported here, were observed at up to 10-fold lower doses in infected than in uninfected mice. The mechanisms responsible for heightened susceptibility under these conditions were evaluated. Kinetic studies demonstrated that virus-infected mice had 3- to 50-fold increases over uninfected mice in peak serum TNF, IL-12, and IFN-gamma levels after LPS administration. All three cytokines contributed to lethality during dual challenge, because neutralization of any one of the factors protected from death. Production of TNF was not dependent on either NK or T cells. In contrast, these populations were the predominant sources of IFN-gamma, as determined by lack of detectable IFN-gamma production in NK and T cell-deficient mice and by intracellular cytokine expression in the cell subsets. Concordant with the demonstrations that both cell populations produced IFN-gamma and that this factor was critical for lethality, removal of either subset alone was not sufficient to protect mice from death resulting from dual challenges. Increased resistance required absence of both cell subsets. Taken together, the data show that during viral infections, the normally protective immune responses can profoundly modify reactions to secondary heterologous challenges, to result in dysregulated cytokine expression and consequent heightened detrimental effects.     

LPS regulation of the immune response: suppression of immune responses to orally administered T-independent antigen

The regulation of immune responses to gastrically administered TI antigens has been investigated, and the characterization of a regulatory cell population has been performed. Intragastric administration of TNP-haptenated homologous erythrocytes (TNP-MRBC) induced splenic IgM anti-TNP PFC responses in LPS nonresponsive C3H/HeJ mice that were higher than those in LPS-responsive C3H/HeN mice and similar to those noted in athymic (nu/nu) C3H/HeN animals. The simultaneous intragastric administration of LPS with TNP-MRBC augmented immune responses in a manner similar to that previously reported for parenterally administered LPS and antigen. Further, LPS-induced augmentation of TNP-MRBC responses was greater in athymic mice. These findings were substantiated using in vitro spleen cultures. Intragastric challenge with a 2nd TI antigen, TNP-LPS, induced approximately 8-fold higher splenic anti-TNP PFC responses in athymic C3H/HeN mice compared with those in euthymic littermates. By admixture of B and T cell populations, it was demonstrated that the host responsiveness to TNP-LPS was negatively regulated by suppressor cells. Suppressive activity resided in a Thy 1.2-bearing, irradiation-resistant, nylon wool-nonadherent cell population. These cells could be demonstrated in spleen and Peyer's patches from young or old LPS-responsive C3H/HeN mice, but not in tissues from LPS nonresponsive C3H/HeJ mice. The specificity of the regulator cells was not limited to TNP-LPS responses, since immune responsiveness to another TI antigen, TNP-dextran, was also under the control of this cell population. These studies confirm the TI nature of TNP-MRBC and indicate that immune responses to gastrically administered antigens such as TNP-LPS, TNP-dextran, and possibly TNP-MRBC are negatively regulated by a suppressor T cell population. A role for endogenous LPS in the generation of regulator cells and the effect of these cells on host responses to gut-derived antigens is discussed.     

Active hexose correlated compound enhances the immune function of mice in the hindlimb-unloading model of spaceflight conditions.

Hindlimb unloading is a ground-based model that simulates some of the aspects of spaceflight conditions, including lack of load bearing on hindlimbs and a fluid shift to the head. It has been shown that treatment with active hexose correlated compound (AHCC) restores resistance to infection in mice maintained under hindlimb-unloading conditions. The present study was designed to clarify the mechanisms by which AHCC enhances resistance to infection in this model. We hypothesized that oral administration of AHCC will enhance the function of the immune system, which could lead to the increased resistance to infection observed in this model. AHCC or the excipient was orally administered to mice, and the function of the immune system was assessed in spleen and peritoneal cells isolated from those groups. The results of the present study showed that administration of AHCC for 1 wk before and throughout the second day of the hindlimb-unloading period enhanced the function of the immune system assessed by spleen cell proliferation and cytokine production in spleens and nitric oxide and cytokine production in peritoneal cells. These findings suggest that AHCC can be used as a potent immunoenhancer, especially in cases in which the immune system is suppressed by any condition, including diseases such as human immunodeficiency virus infection and cancer.