Immunomodulating effects
Many naturally occurring polysaccharides have been reported to be potent immunomodulators.118,119 These polymers can influence innate and cell-mediated immunity through interactions with T cells, monocytes, macrophages, and polymorphonuclear lymphocytes. LBPs have been found to have a variety of immune-modulatory activities in vitro and in vivo.

T cells, B cells, and splenocytes
Chen et al120 compared the immunomodulating effects of different LBP fractions in mice. Crude LBPs isolated from L. barbarum were separated to obtain five homogeneous fractions, namely LBPF1, LBPF2, LBPF3, LBPF4, and LBPF5. The study showed that LBP, LBPF4, and LBPF5 significantly stimulated mouse splenocyte proliferation. The proliferation proved to be of T cells, but not B cells. Cell cycle analysis indicated that LBP, LBPF4, and LBPF5 markedly reduced sub-G1 cells.120 LBP, LBPF4, and LBPF5 activated the transcription factors nuclear factor of activated T-cells (NFAT) and activator protein-1 (AP-1), prompted CD25 (ie, IL-2 receptor-α) expression, and induced IL-2 and interferon (IFN)-γ expressions. NFAT proteins (NFATs 1–5) have crucial roles in the development and function of the immune system. In T cells, NFAT proteins not only regulate activation but are also involved in the control of thymocyte development, T-cell differentiation, and self-tolerance.121 AP-1 regulates gene expression in response to a variety of stimuli, including cytokines, growth factors, stress, and bacterial and viral infections. IL-2 is important for the growth and activation of T cells, and IFN-γ is an important activator of macrophages and inducer of class II major histocompatibility complex (MHC-II) molecule expression. IL-2 is mainly produced by T cells122 and IFN-γ is produced predominantly by NK and NK T cells as part of the innate immune response, and by CD4+ T helper cells (Th1) and CD8+ CTLs once antigen-specific immune response is triggered.123 Administration of LBPs to mice (intraperitoneal [ip] or oral administration [po]) significantly induced T-cell proliferation.120 These results suggest that activation of T lymphocytes by LBPs may contribute to one of their immuno-enhancement functions.
The in vitro and in vivo immunomodulating effects of LBPF4-OL on mouse splenocytes, T cells, B cells, and macrophages were investigated by Zhang et al.124 LBPF4-OL was the glycan part of L. barbarum polysaccharide–protein complex fraction 4 (LBPF4). Splenocytes were stimulated with LBPF4-OL and cytokine concentrations in the supernatants were determined. In the in vivo study, mice were intraperitoneally injected with 100 µg/mL LBPF4-OL daily for 6 days. The results showed that LBPF4-OL markedly induced the splenocyte proliferation, but could not induce proliferation of purified T- and B-lymphocytes.124 B-cell proliferation occurred in the presence of activated macrophages or lipopolysaccharide (LPS). LBPs obviously induced IL-6, IL-8, IL-10, and TNF-α production in splenocytes in a concentration-dependent manner.124 IL-6 is secreted by T cells and macrophages to stimulate immune response during infection and after trauma (especially burns or other tissue damage) leading to inflammation. IL-8 (also called CXCL8 and neutrophil chemotactic factor) is a chemokine produced by macrophages and other cell types such as epithelial cells, airway smooth muscle cells, and endothelial cells. IL-8 induces chemotaxis in target cells (primarily neutrophils but also other granulocytes) toward the site of infection and induces phagocytosis. IL-10 inhibits the production of IFN-γ, IL-2, IL-3, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF) by activated macrophages and helper T cells. TNF-α is mainly produced by activated macrophages (M1), although it can be produced by many other cell types such as CD4+ T lymphocytes, monocytes, NK cells, neutrophils, mast cells, eosinophils, and neurons.125 The primary role of TNF-α is in the regulation of immune cells. TNF-α induces fever, apoptotic cell death, cachexia, and inflammation; inhibits tumorigenesis and viral replication; and responds to sepsis via IL-1 and IL-6 producing cells.125 Flow cytometer analysis showed that LBPF4-OL prompted CD86 (B7-2) and MHC-II molecule expression on macrophages and greatly promoted release of TNF-α and IL-1β from macrophages.124 IL-1β is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1. This cytokine is an important mediator of the inflammatory response and is involved in a variety of cellular activities, including cell proliferation, differentiation, and apoptosis.
Vidal et al126 revealed that dietary wolfberry supplementation enhanced both in vivo (delayed-type hypersensitivity) and ex vivo (T-cell proliferation) T-cell response to specific antigens, but it did not affect mitogen-induced T-cell or B-cell proliferation in young and aged mice. Over 44 days, young-adult (2 months) and aged (21 months) C57BL/6J mice were fed ad libitum with a controlled diet and received drinking water supplemented or not with 0.5% (wt/vol) Lacto-Wolfberry. All mice were immunized on day 15 and challenged on day 22 with a T-cell-dependent antigen, keyhole limpet hemocyanin. The study showed that Lacto-Wolfberry supplementation significantly increased in vivo systemic immune markers.126 Both antigen-specific humoral response and cell-mediated immune responses in young-adult and aged mice were enhanced. However, no significant effect of Lacto-Wolfberry supplementation was observed on ex vivo splenocyte proliferative response to mitogens and on splenocyte T-cell subsets.126 These data suggest that dietary intake of Lacto-Wolfberry may favorably modulate the poor responsiveness to antigenic challenge observed with aging.
Zhang et al127 further compared the effect of LBPF4 and LBPF4-OL on the proliferation of splenocytes and mitogen-induced B and T lymphocytes in female Balb/C mice. LBPF4 and LBPF4-OL were isolated in the fruit bodies of L. barbarum through a series of diethylaminoethyl anion exchange cellulose and gel-permeation chromatography. The molecular weight of LBPF4 was 214.8 kDa, and consisting of 17 amino acids and four kinds of monosaccharides. The molecular weight of LBPF4-OL was 181 kDa, consisting of three types of monosaccharides.127 The effects on cytokine secretion, the phagocytic potential of macrophages, and the expression level of intracellular signaling molecules including NF-κB and B-cell-specific activator protein (BSAP, also named Pax5) were also determined. BSAP/Pax5 is essential for commitment of lymphoid progenitors to the B lymphocyte lineage.128 Spleen cells (5×105) were stimulated with 10 µg/mL, 50 µg/mL, and 100 µg/mL LBPF4-OL. Concanavalin A (Con A, 0.5 µg/mL) and LPS (5 µg/mL) were included as positive controls for the proliferation of T and B cells, respectively. The results showed that 50 µg/mL LBPF4 significantly enhanced spleen cell proliferation ∼3.2 fold, while LBPF4-OL enhanced proliferation 7.2 fold. Administration of 10 µg/mL, 50 µg/mL, or 100 µg/mL LBPF4 but not LBPF4-OL, significantly enhanced the Con A-induced T lymphocyte proliferation.127 However, LPS-induced B-cell proliferation was enhanced by 10 µg/mL, 50 µg/mL, or 100 µg/mL of both LBPF4 and LBPF4-OL. Administration of 50 µg/mL LBPF4-OL was more effective on inducing the proliferation of splenocytes and LPS-stimulated B cells than 100 µg/mL LBPF4. LBPF4 appeared to induce lymphocyte proliferation predominantly depending on both B and T cells, and LBPF4-OL induced lymphocytes proliferation only depending on B cells. The stimulation of murine peritoneal macrophages with LBPF4 and LBPF4-OL resulted in a comparable dose-dependent increase of the production of TNF-α and IL-1β.127 In addition, both LBPF4 and LBPF4-OL at concentrations of 10 µg/mL, 50 µg/mL, and 100 µg/mL increased the secretion of NO to comparable levels. Administration of 10 µg/mL LBPF4 and LBPF4-OL showed no significant effects on the phagocytic activity of resting macrophages in mice, but the macrophage chicken erythrocyte phagocytic activity was significantly increased by low concentrations of LBPF4 and LBPF4-OL. About 50 µg/mL (but not 10 µg/mL) LBPF4 and LBPF4-OL significantly promoted BSAP and NF-κB activity.127 These data suggest that LBPF4-OL can only enhance B cell and macrophage functions, but polysaccharide–protein complex LBPF4 can enhance the function of both T and B cells and macrophages.
Recently, Zhang et al129 found that LBPF4-OL acted as an activator of the Toll-like receptor 4 (TLR4)/p38 MAPK signaling pathway using TLR4 knockout mice. LBPF4-OL significantly induced TNF-α and IL-1β production in peritoneal macrophages isolated from wild type (C3H/HeN) but not TLR4-deficient mice (C3H/HeJ). The proliferation of LBPF4-OL-stimulated lymphocytes from C3H/HeJ mice was significantly lower than that of lymphocytes from C3H/HeN mice.129 Furthermore, through a bio-layer interferometry assay, LPS but not LBPF4-OL directly associated with the TLR4/MD2 molecular complex. Flow cytometry analysis indicated that LBPF4-OL markedly upregulated TLR4/MD2 expression in both peritoneal macrophages and Raw264.7 cells.129 LBPF4-OL also increased the phosphorylation of p38-MAPK and inhibited the phosphorylation of JNK and Erk1/2. These data suggest that LBPF4-OL can activate TLR4/p38 MAPK signaling pathway.

Peripheral blood mononuclear cells
IL-2 is necessary for the growth, proliferation, and differentiation of T lymphocytes to become functional T cells.122 Antigen binding to the T-cell receptor stimulates the secretion of IL-2 by T cells and the expression of IL-2 receptors (IL-2Rs). The IL-2/IL-2R interaction stimulates the growth, differentiation, and survival of antigen-specific CD4+ and CD8+ T cells. IL-2 plays an important role for the development of T-cell-dependent immune memory. An in vitro study reported the effects of LBPs on the expression of IL-2 and TNF-α in human peripheral blood mononuclear cells (PBMCs) from healthy volunteers.130 The LBPs used in this study were the third fraction of LBPs extracted with hot water from L. barbarum planted in Zhongning, Ningxia, People’s Republic of China and isolated by anionic exchange chromatography and gel-filtration chromatography. Administration of 10 mg/L LBPs increased the expression of IL-2 and TNF-α at both mRNA and protein levels in a dose-dependent manner. Treatment of human PBMCs with 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L LBPs increased IL-2 mRNA 1.8-, 3.9-, 7.0-, and 7.4-fold, respectively. The activity of IL-2 was increased 4.3-, 7.7-, 14.2-, and 16.0-fold, respectively, compared to the negative control.130 Treatment of PBMCs with 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L LBPs increased TNF-α mRNA level 2.4-, 3.9-, 6.1-, and 15.4-fold, respectively. The activity of TNF-α after treatment with 5 mg/L, 10 mg/L, 20 mg/L, and 40 mg/L LBPs for 8 hours was increased 7.1-, 9.1-, 13.6-, and 15.2-fold, respectively, compared to the negative control. LBPs may induce immune responses that contribute to the therapeutic effect in cancer.

Macrophages
Macrophages play a crucial role in innate immunity and also help initiate adaptive immunity.131,132 Macrophages predominantly expressing the killer phenotype are called M1 macrophages, whereas those involved in tissue repair are called M2 macrophages.133 The primary role of macrophages is to phagocytose or engulf and then digest cellular debris and pathogens; they also stimulate lymphocytes and other immune cells to respond to pathogens. M1 macrophages are activated by LPS and IFN-γ and secrete high levels of IL-12 and low levels of IL-10; and M2 macrophages produce high levels of IL-10, TGF-β, and low levels of IL-12.133 IL-12 is involved in the stimulation and maintenance of Th1 cellular immune responses and also has an important role in enhancing the cytotoxic function of NKs. Macrophages can be identified by specific expression of a number of proteins including CD14, CD40, CD11b, CD64, F4/80 (mice)/EMR1 (human), lysozyme M, MAC-1/MAC-3, and CD68. LBPs are able to activate macrophages. A study found that LBPs (50 mg/kg, ip) markedly upregulated the expressions of CD40, CD80 (B-lymphocyte activation antigen B7-1), CD86 (B-lymphocyte activation antigen B7-2), and MHC-II molecules on peritoneal macrophages. In vitro studies showed that LBPs activated transcription factors NF-κB and AP-1, induced TNF-α, IL-1β, and IL-12p40 mRNA expression, and enhanced TNF-α production in RAW264.7 macrophage cells in a dose-dependent manner.134 Furthermore, LBPs significantly enhanced macrophage endocytic and phagocytic capacities in vivo. These results indicated that LBPs enhance innate immunity by activating macrophages. The mechanism might be through activation of transcription factors NF-κB and AP-1 to induce TNF-α production and upregulation of MHC-II co-stimulatory molecules.134
An in vitro study by Teng et al135 investigated the inhibitory effects of LBPs on the production of LPS-induced proinflammatory mediators in BV2 microglia. The data showed that LPS induced the activation of NF-κB and its upstream protein caspase 3. NF-κB plays a key role in inflammatory disease and may be involved in autophagy, while autophagy itself may also participate in the pathogenesis of inflammation and inflammatory disease.136 LPS also unregulated the expression of an additional apoptosis-inducing factor with a passive role in the maturation of caspase processing, HSP60, in BV2 microglial cells and increased the release of TNF-α and HSP60 in the culture media. Following treatment with LBPs, the activated caspase 3 were significantly suppressed. Furthermore, the enhanced expression of HSP60 was reduced and the LPS-induced release of TNF-α and HSP60 was inhibited. These results suggest that LBPs may have therapeutic potential for the treatment of neurodegenerative diseases that are accompanied by microglial activation.
Peng et al137 investigated the effect of Lycium ruthenicum polysaccharides (LRGP3) on inflammatory reactions induced by LPS in mouse macrophage RAW264.7 cells. The results showed that LRGP3 treatment significantly inhibited the LPS-induced NO production and the mRNA expression of iNOS, as well as the level of TLR4. Furthermore, LRGP3 treatment prevented IκBα degradation and reduced phospho-NF-κB p65 protein expression in LPS-stimulated RAW264.7 cells.137 Meanwhile, the levels of proinflammatory cytokines, such as IL-1α, IL-6, and TNF-α, were suppressed by LRGP3 in LPS-stimulated RAW264.7 cells. LRGP3 attenuated LPS-induced inflammation via inhibiting TLR4/NF-κB signaling pathway.

Natural killers
NK cells are major effectors of the innate immunity, providing rapid responses to virally infected cells and respond to tumor formation.138,139 Cytokines involved in NK activation include IL-12, IL-15, IL-18, IL-2, and CCL5. NK cells are activated in response to IFNs or macrophage-derived cytokines. NK cells control viral infections by secreting IFN-γ and TNFα.138,139 IFN-γ activates macrophages for phagocytosis and lysis, and TNFα acts to promote direct NK tumor cell killing. NKs express the surface markers CD16 (FcγRIII) and CD56 in humans. A recent study by Huyan et al140 reported the effects of LBPs on primary human NK cells under normal or simulated microgravity conditions. The results demonstrated that LBPs markedly promoted the cytotoxicity of NK cells by enhancing IFN-γ and perforin secretion and increasing the expression of the activating receptor NKp30 under normal conditions. Meanwhile LBPs enhanced NK cell function under simulated microgravity conditions by restoring the expression of the activating receptor NKG2D and reducing the early apoptosis and late apoptosis/necrosis.140 In addition, the antibody neutralization test showed that the complement receptor CR3 may be the critical receptor involved in LBP-induced NK cells activation. These findings indicate that LBPs are potent immune regulators and can promote the immune functions of the public and astronauts during space missions.

Dendritic cells
Dendritic cells (DCs) are potent antigen-presenting cells that play pivotal roles in the initiation of the adaptive (T and B cell) immune response.141,142 The principal function of DCs is to present antigens, and only DCs have the ability to induce a primary immune response in resting naïve T lymphocytes. DCs also play a role in the maintenance of B cell function and recall responses. DCs express a variety of adhesion molecules including CD11a (integrin lymphocyte function-associated antigen-1, namely LFA-1), CD11c/CD18, CD50 (ICAM-2), CD54 (ICAM-1), CD58 (LFA-3), and CD102 (ICAM-3).141,142 CD11a/LFA-1 plays a central role in leukocyte intercellular adhesion through interactions with its ligands, ICAMs 1-3, and also functions in lymphocyte costimulatory signaling. DCs also express costimulatory molecules including CD80 (B7-1) and CD86 (B7-2), which are upregulated during DC activation. CD86 tends to be a marker of early DC maturation, while CD80 only appears in mature DC.141,142
The effects of LBPs on the phenotypic and functional maturation of murine bone marrow DCs (BMDCs) were investigated in vitro by Zhu et al.143 The co-expression of MHC-II, CD11c, and secretion of IL-12 p40 by BMDCs stimulated with 100 mg/L LBPs increased significantly. LBPs are capable of promoting both the phenotypic and functional maturation of murine BMDCs in vitro.
Chen et al144 reported that LBPs induced phenotypic and functional maturation of DCs with strong immunogenicity. LBPs can upregulate the expression of CD40, CD80, CD86, and MHC-II molecules on DCs, downregulate DC uptake of antigen, enhance allostimulatory activity of DCs, and induce the production of IL-12p40 and p70 in DCs.144 LBP-treated DCs can enhance both Th1 and Th2 responses in vitro and in vivo. LBPs may serve as a potent adjuvant for the design of DC-based vaccines.
Chen et al145 investigated the effect of LBPs on differentiation and maturation of healthy human peripheral blood-derived DCs cultured in different tumor microenvironment in vitro. Peripheral blood-derived DC precursor cells were obtained by the density-gradient centrifugation method, and the tumor-cell supernatants were used to prepare conditioned medium. The GM-CSF and IL-4-induced DC precursor cell differentiation to DCs, the TNF-α promoted the immature DCs developed to mature DCs. In LBP-treated groups, the molecular phenotype of DCs, their capacity to stimulate allogeneic lymphocyte proliferation, and the levels of IL-12p70 and IFN-γ secretion were higher than the untreated group.145 Meanwhile, the expression of NF-κB of the DCs in the medium treated with LBPs was higher than the untreated group.145 Between the two different tumor microenvironment groups, the nuclear NF-κB expression was obviously different. LBP could increase the expression of the phenotype of DCs via NF-κB signaling pathway.

Follicular helper T cells
Follicular helper T (Tfh) cells are recognized as a subset of helper T cells that regulate the multiple stages of B-cell maturation and function.146 Tfh cells retain intense expression of CXCR5, which directs these cells toward CXCL13-rich areas within the germinal center. Tfh cells express a number of costimulatory molecules, such as inducible costimulator and CD40L that have the capacity to restrain their interaction with B cells and antigen-presenting cells, including CTLA-4 and PD-1, which may reflect their discriminating role in the germinal center. Tfh cells also express a number of cytokines that facilitate antibody production including IL-4, IL-10, and IL-21.
A recent study by Su et al147 reported that LBPs were able to activate CXCR5+/PD-1+ Tfh cells and induced IL-21 secretion in female Balb/C mice. Mice were immunized once with ip injection of 0.2 mL of 108 TCID50 rAd5VP1. LBPs were given to mice daily for 7 days by gastric gavage at 5 mg/kg, 25 mg/kg, or 50 mg/kg body weight. After 7 days, mice were sacrificed, the splenocytes were harvested, and the number of CXCR5+/PD-1+ Tfh cells was determined by three-color flow cytometry.147 Mouse splenocytes were also analyzed by flow cytometry to determine the counts of B220+/GL-7+ B cells. The study showed that LBP treatment increased the percentage of CXCR5+/PD-1+ Tfh cells within total CD4+ T cells; 5 mg/kg (2.17%±0.07%), 25 mg/kg (3.93%±0.74%), and 50 mg/kg (3.84%±0.20%). Administration of 5 mg/kg LBP for 7 days exhibited a minor effect on the production of IL-21, whereas 25 mg/kg and 50 mg/kg LBPs significantly increased the production of IL-21 when compared with mice treated with phosphate buffered saline only.147 LBPs also promoted the formation of germinal centers and production of B220+/GL-7+ germinal center B cells in mice. The fraction of B220+/GL-7+ B cells was significantly increased with 25 mg/kg and 50 mg/kg LBPs compared with mice receiving phosphate buffered saline only (1.80%±0.49%). Moreover, LBPs as an adjuvant increased generation of rAd5VP1-induced Tfh cells in mice.147 There was a marked increase in the number of CXCR5+/PD-1+/CD4+ Tfh cells and B220+/GL-7+ germinal center B cells in mice immunized with 108 TCID50 rAd5VP1 plus LBPs. These results indicate that LBPs may enhance T-cell-dependent antibody responses by acting as an adjuvant for the generation of Tfh cells.

LBP as a vaccine adjuvant
LBPs stimulated moderate immune responses, and therefore, could potentially be used as a substitute for oil adjuvants in vaccines. Subfractions of polysaccharides, 12.5 mg/kg, 25 mg/kg, or 50 mg/kg LBP3a, were mixed with a DNA vaccine encoding the major outer membrane protein of Chlamydophila abortus.148 Balb/C mice were inoculated at days 0, 14, and 28, and challenged on day 44. Serum antibody levels, in vitro lymphocyte proliferation, the levels of IL-2, IFN-γ, and TNF-α, and Chlamydia clearance in the spleen were monitored. A combination of DNA vaccine plus LBP3a induced significantly higher antibody levels in mice, higher T-cell proliferation, and higher levels of IFN-γ and IL-2. Mice immunized with DNA vaccine and LBPs showed significantly higher levels of Th1 immune response and Chlamydia clearance in mouse spleen. The immunoenhancing effect induced by 25 mg/kg LBP3a was more effective than that induced by 12.5 mg/kg and 50 mg/kg LBP3a. These results suggest that LBPs may be used as an effective adjuvant with a DNA vaccine against swine C. abortus.
Vaccination is the most efficient strategy to prevent influenza infection. However, vaccine efficacy is significantly diminished in the elderly due to the age-related impairment of both innate and adaptive immune responses. A recent study149 has examined whether dietary wolfberry supplementation enhanced the protective effect of influenza vaccine against influenza challenge in aged male C57BL/6J mice (20–22 months old). The mice were fed a 5% milk-based preparation of wolfberry (Nestec) or fed with 5% corn starch (control) for 30 days, then immunized with an influenza vaccine or saline (control) by ip injection on days 31 and 52 of the dietary intervention, and finally challenged with influenza A/Puerto Rico/8/34 virus (Sino Biological, Bejing, People’s Republic of China) with an aluminum adjuvant at a ratio of 1:1. The milk-based preparation of wolfberry contained 530 mg/g of wolfberry fruit, 290 mg/g of skimmed milk, and 180 mg/g of maltodextrin. At day 73, mice were infected with influenza A/Puerto Rico/8/34 virus and were monitored daily for weight loss and mortality.149 Mice fed with wolfberry diet had higher influenza IgG titers, less weight loss, and improved survival rate in influenza-infected mice when compared with the mice treated by influenza vaccine alone.149 Furthermore, an in vitro study showed that administration of 100 mg/L, 200 mg/L, 400 mg/L, or 800 mg/L wolfberry supplementation enhanced maturation and activity of antigen-presenting DCs isolated from the bone marrow of aged mice. Wolfberry extract dose-dependently increased the percentages of DCs expressing MHC-II and T-cell costimulatory molecules CD40, CD80, and CD86 and their expression. Wolfberry enhanced the production of proinflammatory IL-12 and TNF-α.149 With improved maturation of DCs, the endocytic capability of DCs was significantly reduced when treated with wolfberry extract. Adoptive transfer of wolfberry-treated bone marrow DCs loaded with ovalbumin323–339 to recipient mice promoted antigen-specific T-cell proliferation as well as IL-4 and IFN-γ production in CD4+ T cells.149 Wolfberry may enhance the antigen-presenting function of DCs, leading to a higher level of antigen-specific T-cell effector function involving at least Th1 and Th2 responses. These data indicate that dietary wolfberry potentiates the efficacy of influenza vaccination, resulting in better host protection to prevent subsequent influenza infection via improved DC function.

Clinical studies
Amagase et al150 investigated the systematic effects of consuming 120 mL/day GoChi for 30 days on immune function, general well-being, and safety in a randomized, double-blind, placebo-controlled clinical study in 60 older Chinese healthy adults (55–72 years old). The GoChi group showed a statistically significant increase in the number of lymphocytes and levels of IL-2 and IgG compared to pre-intervention and the placebo group, whereas the number of CD4, CD8, and NK cells or levels of IL-4 and IgA were not significantly altered. The placebo group showed no significant changes in any immune measures, whereas the GoChi group showed a significant increase in general feelings of well-being, such as fatigue and sleep, and showed a tendency for increased short-term memory and focus between pre- and post-intervention; the placebo group showed no significant positive changes in these measures.150 GoChi was well tolerated. No adverse reactions, abnormal symptoms, or changes in body weight, blood pressure, pulse, visual acuity, urine, stool, or blood biochemistry were noted in either group.150 Daily consumption of GoChi significantly increased several immunological responses and subjective feelings of general well-being without any adverse reactions in the elderly.
A recent study by Vidal et al151 reported that elderly persons who consumed Lacto-Wolfberry for 3 months (13.7 g/day in the form of the same milk-based preparation of wolfberry) had higher serum influenza-specific IgG concentrations and seroconversion rate after receiving an influenza vaccine compared with age-matched elderly individuals in the placebo group. The study was conducted in 150 healthy community-dwelling Chinese elderly (65–70 years old) supplemented with Lacto-Wolfberry or placebo (13.7 g/day). No serious adverse reactions were reported during the trial, neither symptoms of influenza-like infection nor changes in body weight and blood pressure, blood chemistry or cells composition, and autoantibodies levels were observed.151 Lacto-Wolfberry supplementation had no significant effect on delayed-type hypersensitivity response and inflammatory markers. These data show that chronic dietary supplementation with Lacto-Wolfberry in the elderly enhances their capacity to respond to influenza vaccine challenge.

Summary of immunomodulating effects of LBPs
A number of in vitro and in vitro studies have revealed the immunomodulating activities of LBPs (Figure 9). LBPs promote the proliferation and activity of splenocytes, T cells, B cells, macrophages, and NK cells. LBPs induce IL-6, IL-8, IL-10, and TNF-α production in splenocytes. LBPs stimulate PBMCs to produce IL-2 and TNF-α. IL-2 stimulates growth and differentiation of T cells. LBPs promote T lymphocytes and macrophages to release important cytokines such as IL-10 and TNF-α. LBPs activate macrophages and upregulate the expressions of CD40, CD80, CD86, and MHC-II molecules. LBPs activate transcription factors NF-κB and AP-1, induce TNF-α, IL-1β, and IL-12p40 expression in macrophages. LBPs significantly enhance macrophage endocytic and phagocytic capacities. LBPs promote the cytotoxicity of NK cells by enhancing IFN-γ and perforin release and the expression of the activating receptors NKp30 and NKG2D. LBPs also stimulate macrophages and NK cells to release TNF-α and IL-1β. LBPs activate the transcription factors NFAT and AP-1 and prompt CD25 (IL-2 receptor-α) expressions. LBPs induce the maturation of DCs and improve their antigen-presenting function. LBPs can upregulate the expression of CD40, CD80, CD86, and MHC-II molecules in bone marrow- and peripheral blood-derived DCs, downregulate DC uptake of antigen (Ag), enhance allostimulatory activity of DCs, and induce the production of IL-12p40 and p70 in DCs. LBP-treated DCs can enhance both Th1 and Th2 responses. LBPs potentiate the immune responses of DNA vaccine against C. abortus in mice. LBPs activate CXCR5+PD-1+ Tfh cells and induce IL-21 secretion. Dietary wolfberry supplementation enhances both in vivo and ex vivo T-cell response to specific antigens. Elderly persons who consume Lacto-Wolfberry for 3 months show higher serum influenza-specific IgG concentrations and seroconversion rate after receiving an influenza vaccine.