β-glucans are one of the most abundant forms of polysaccharides found inside the cell wall of bacteria and fungus. All β-glucans are glucose polymers linked together by a 1→ 3 linear β-glycosidic chain core and they differ from each other by their length and branching structures 14 (Figure 1). The branches derived from the glycosidic chain core are highly variable and the 2 main groups of branching are 1→4 or 1→6 glycosidic chains. These branching assignments appear to be species specific, for example, β-glucans of fungus have 1→6 side branches whereas those of bacteria have 1→4 side branches. The alignments of branching follow a particular ratio and branches can arise from branches (secondary branches). In aqueous solution, β-glucans undergo conformational change into triple helix, single helix or random coils. The immune functions of β-glucans are apparently dependent on their conformational complexity 15. It has been suggested that higher degree of structural complexity is associated with more potent immunmodulatory and anti-cancer effects.

For research purposes, the composition or structural information of β-glucans can be evaluated by a variety of methods including liquid chromatography/mass spectrometry (LC/MS)16, high performance liquid chromatography (HPLC)17 and less often X-ray crystallography 18 or atomic force microscopy 19. However, due to the tedious and lack of quantitative nature of most of these technical methods, they cannot be applied routinely as a screening tool. Other less sophisticated techniques in studying the β-glucans contents include phenol-sulphuric acid carbohydrate assay, aniline blue staining method and ELISA. Because chemical modification invariably induces changes in the natural conformation, most of these methods cannot reflect the genuine relationship between the structure and the bioactivity. Among them, aniline blue staining method is a relatively simple method to screen for β-glucan because of its ability to retain the natural conformation of β-glucans during the staining process. It also has a good specificity for β-glucans but its limitation is that it can only measure the core 1→3 linear glycosidic chain and not the branches.

Endotoxin contamination is another important issue affecting the safety and potential biological effect of β-glucan. Lipopolysaccharide (LPS) is an endotoxin found inside the Gram negative bacterial cell wall and consists of three main parts including lipid A, core and polysaccharide chain 20. Among them, lipid A was found to be the major component that can initiate an immune response. LPS contamination can occur during the culture or preparation of β-glucans. Since LPS is one of the most potent immune stimulator and its contamination can lead to false positive results in immune tests, quantification of LPS should be performed, which can be evaluated by either the rabbit pyrogen test or the modified limulus amebocyte lysate (LAL) assay with devoid factor G 21.

Most β-glucans are considered as non-digestible carbohydrates and are fermented to various degrees by the intestinal microbial flora 222324. Therefore, it has been speculated that their immunomodulatory properties may be partly attributed to a microbial dependent effect. However, β-glucans in fact can directly bind to specific receptors of immune cells, suggesting a microbial independent immunomodulatory effect 25. The pharmacodynamics and pharmacokinetics of β-glucans have been studied in animal and human models.

Current data suggests that β-glucans are potent immunomodulators with effects on both innate and adaptive immunity. The ability of the innate immune system to quickly recognize and respond to an invading pathogen is essential for controlling infection. Dectin-1, which is a type II transmembrane protein receptor that binds β-1,3 and β-1,6 glucans, can initiate and regulate the innate immune response 313233. It recognizes β-glucans found in the bacterial or fungal cell wall with the advantage that β-glucans are absent in human cells. It then triggers effective immune responses including phagocytosis and proinflammatory factors production, leading to the elimination of infectious agents 3435. Dectin-1 is expressed on cells responsible for innate immune response and has been found in macrophages, neutrophils, and dendritic cells 36. The Dectin-1 cytoplasmic tail contains an immunoreceptor tyrosine based activation motif (ITAM) that signals through the tyrosine kinase in collaboration with Toll-like receptors 2 and 6 (TLR-2/6) 343738. The entire signaling pathway downstream to dectin-1 activation has not yet been fully mapped out but several signaling molecules have been reported to be involved. They are NF-κB (through Syk-mediate pathway), signaling adaptor protein CARD9 and nuclear factor of activated T cells (NFAT) 394041 (Fig. 3). This will eventually lead to the release of cytokines including interleukin (IL)-12, IL-6, tumor necrosis factor (TNF)-α, and IL-10. Some of these cytokines may play important role in the cancer therapy. On the other hand, the dendritic cell-specific ICAM-3-grabbing non-integrin homolog, SIGN-related 1 (SIGNR1) is another major mannose receptor on macrophages that cooperates with the Dectin-1 in non-opsonic recognition of β-glucans for phagocytosis 42 (Fig 3). Furthermore, it was found that blocking of TLR-4 can inhibit the production of IL-12 p40 and IL-10 induced by purified Ganoderma glucans (PS-G), suggesting a vital role of TLR-4 signaling in glucan induced dendritic cells maturation. Such effect is also operated via the augmentation of the IκB kinase, NF-κB activity and MAPK phosphorylation 43. One additional point to note is that those studies implied the interaction between β-glucans and TLR all used non-purified β-glucans, therefore the actual involvement of pure β-glucans and TLR remains to be proven.

Other possible receptors and signaling pathways induced by β-glucans are less definite at the moment. For example, lentinan, a form of mushroom derived β-glucans, has been found to bind to scavenger receptor found on the surface of myeloid cells and triggers phosphatidylinositol-3 kinase (PI3K), Akt kinase and p38 mitogen-activated protein kinase (MAPK) signaling pathway 44(Fig. 3). But no specific β-glucans scavenger receptor has been identified so far. Candida albicans derived β-glucans but not other forms of pathogenic fungal β-glucans can bind to LacCer receptor and activate the PI-3K pathway in controlling the neutrophil migration 45 (Fig. 3), but such activation pathway may involve other molecules found in the Candida derived β-glucans.

We found that β-glucans can induce human peripheral blood mononuclear cells proliferation 46. It can also enhance phenotypic and functional maturation of monocyte derived dendritic cells with significant IL-12 and IL-10 production. Similar findings were found by Lin et al. using PS-G, in addition, treatment of dendritic cells with PS-G resulted in enhanced T cell-stimulatory capacity and increased T cell secretion of interferon-γ and IL-10 4347. This action is at least mediated in part through the Dectin-1 receptor. The potency of such immunomodulating effects differs among β-glucans and purified polysaccharides of different size and branching complexity. In general, bigger size and more complex β-glucans such as those derived from Ganoderma lucidum have higher immunomodulating potency.

The adaptive immune system functions through the combined action of antigen-presenting cells and T cells. Specifically, class I major histocompatibility complex (MHC-I) antigen presentation to CD8(+) cytotoxic T cells is limited to proteosome-generated peptides from intracellular pathogens. On the other hand, the class II MHC (MHC-II) endocytic pathway presents only proteolytic peptides from extracellular pathogens to CD4(+) T helper cells. Carbohydrates have been previously thought to stimulate immune responses independently of T cells 48. However, zwitterionic polysaccharides (polysaccharides that carry both positive and negative charges) such as β-glucans can activate CD4(+) T cells through the MHC-II endocytic pathway 49. β-glucans are processed to low molecular weight carbohydrates by a nitric oxide-mediated mechanism. These carbohydrates will then bind to MHC-II inside antigen-presenting cells such as dendritic cells for presentation to T helper cells. Initial data suggested that it subsequently leads to Th-1 response, but there are conflicting data related to this aspect. In our in vitro data, β-glucans do not tend to polarize T cells into either Th-1, Th-2 or regulatory T cells 46. However, recent publications suggested β-glucans such as zymosan may induce T-cells into T-reg cells in a NOD mice model 50. Therefore, whether β-glucans can induce important immunologic responses through T cell activation remain to be further investigated.

Another mechanism of β-glucan action is mediated via the activated complement receptor 3 (CR3, also known as CD11b/CD18), which is found on natural killer (NK) cells, neutrophils, and lymphocytes. This pathway is responsible for opsonic recognition of β-glucans leading to phagocytosis and reactor cells lysis. β-glucans bind to the lectin domain of CR3 and prime it for binding to inactivated complement 3b (iC3b) on the surface of reactor cells. The reactor cells can be of any cell type including cancer cells tagged with monoclonal antibody and coated with iC3b. The β-glucans-activated circulating cells such as the CR3 containing neutrophils will then trigger cell lysis on iC3b-coated tumor cells 28. Similarly, majority of the human NK cells express CR3 and it was shown that opsonization of NK cells coated with iC3b leads to an increase in the lysis of the target. The beta chain of the CR3 molecule (CD18) rather than the alpha chain (CD11b) is responsible to the β-glucan binding 5152.

This concept was supported by in vivo study demonstrating barley β-1,3;1,4-glucan given orally can potentiate the activity of an antitumor monoclonal antibody (anti-ganglioside-2 or "3F8"), leading to enhanced tumor regression and survival on a human neuroblastoma xenografts mouse model 53. 3F8 plus β-glucan was shown to produce near-complete tumor regression or disease stabilization whereas 3F8 or β-glucan alone showed no significant effect. The median survival of the 3F8 plus β-glucan group was 5.5-fold higher than that of the control groups, and up to 47% of the mice remained progression free in contrast to <3% of controls at the end of the study period. No toxicities were noted in all mice treated with β-glucan, 3F8, or 3F8 plus β-glucan.

A similar xenograft model was adopted subsequently for investigating various targeted tumor antigens and tumor types. It was found that β-glucan exerts similar anti-tumor effects irrespective of antigens (GD2, GD3, CD20, epidermal growth factor-receptor, and HER-2) or human tumor types (neuroblastoma, melanoma, lymphoma, epidermoid carcinoma, and breast carcinoma) or tumor sites (subcutaneous versus systemic). The effect was correlated with the molecular size of the β-1,3;1,4-glucan 5354.

Furthermore, 2 other receptors known as scavenger 55 and lactosylceramide 5657 also bind β-glucans and can elicit a range of responses. β-glucans can enhance endotoxin clearance via scavenger receptors by decreasing TNF production leading to improved survival in rats subjected to Escherichia coli sepsis 58. On the other hand, β-glucans binding to lactosylceramide receptor can enhance myeloid progenitor proliferation and neutrophil oxidative burst response, leading to an increase in leukocyte anti-microbial activity. It is also associated with the activation of NF-κB in human neutrophils 59. Again in other studies, structurally different β-glucans appear to have different affinity toward these receptors. For example, only high molecular weight β-glucans can effectively bind to the lactosylceramide receptor. Therefore, markedly different host responses induced by different β-glucans are expected.

In summary, β-glucans act on a diversity of immune related receptors in particularly Dectin-1 and CR3, and can trigger a wide spectrum of immune responses. The targeted immune cells of β-glucans include macrophages, neutrophils, monocytes, NK cells and dendritic cells (Figure 3). The immunomodulatory functions induced by β-glucans involve both innate and adaptive immune response. β-glucans also enhance opsonic and non-opsonic phagocytosis. Whether β-glucans polarize the T cells subset towards a particular direction remains to be explored.

It is becoming clear that β-glucans themselves have no direct cytotoxic effects. Studies implicating the cytotoxic effects of β-glucans were either from studies using crude extracts of β-glucan containing herbs or the use of β-glucan primed monocytes. For β-glucan containing herbs like Ganoderma lucidum (Lingzhi), there are other active components such as ganoderic acid from its mycelium 60 and triterpenes from its spore 616263, which have all been shown to have direct anti-cancer effects independently. We did not find any direct cytotoxic effects of β-glucans on a panel of common cancer cell lines tested including carcinoma, sarcoma, and blastoma. β-glucans also did not trigger any apoptotic pathways and had no direct effect on the telomerase and telomeric length of the cancer cells (unpublished data). In contrast, it stimulated the proliferation of monocytic lineage leukemic cells in-vitro and can facilitate the maturation of dendritic cells derived from leukaemic cells 64. Hence, whether it is beneficial to apply β-glucans on leukemic patients remains controversial and has to be considered with caution.

In the English literature, there are no clinical trials that directly assessed the anti-cancer effects of purified β-glucans in cancer patients. Most studies were assessing the toxicity profile or underlying immune changes of the cancer patients without addressing on the change of cancer status. In addition, most of the related studies used either crude herbal extracts or a fraction of the extracts instead of purified β-glucans. Therefore, it is difficult to identify whether the actual effects were related to β-glucans or other confounding chemicals found in the mixture.

In a prospective clinical trial of short term immune effects of oral β-glucan in patients with advanced breast cancer, 23 female patients with advanced breast cancer were compared with 16 healthy females control 65. Oral β-1,3;1,6-glucan was taken daily. Blood samples were recollected on the day 0 and 15. It was found that despite a relatively low initial white cell count, oral β-glucan can stimulate proliferation and activation of peripheral blood monocytes in patients with advanced breast cancer. Whether that can be translated into clinical benefit remains unanswered.

Many edible fungi particularly in the mushroom species yield immunogenic substances with potential anticancer activity 66. β-glucans are one of the common active components (Table 1). In limited clinical trials on human cancers, most were well tolerated. Among them, lentinan derived from Lentinus edodes is a form of β-glucans 67. Since it has poor enteric absorption, intrapleural, intra-peritoneal 68 or intravenous routes had been adopted in clinical trials which showed some clinical benefit when used as an adjuvant to chemotherapy 69. Schizophyllan (SPG) or sizofiran is another β-glucan derived from Schizophyllan commune. Its triple helical complex β-glucans structure prevents it from adequate oral absorption so an intratumoral route or injection to regional lymph nodes had been adopted 7071. In a randomized trial, SPG combined with conventional chemotherapy improved the long term survival rate of patients with ovarian cancer 72. But whether the prolonged survival can subsequently led to a better cure rate remain unanswered.

Maitake D-Fraction extracted from Grifola frondosa (Maitake mushroom) was found to decrease the size of the lung, liver and breast tumors in >60% of patients when it was combined with chemotherapy in a 2 arms control study comparing with chemotherapy alone 73. The effects were less obvious with leukemia, stomach and brain cancer patients 74. But the validity of the clinical study was subsequently questioned by another independent observer 75. Two proteoglycans from Coriolus versicolor (Yun Zhi) – PSK (Polysaccharide-K) and PSP (Polysaccharopeptide) – are among the most extensively studied β-glucan containing herbs with clinical trials information. However, both PSK and PSP are protein-bound polysaccharides, so their actions are not necessary directly equivalent to pure β-glucans 76. In a series of trials from Japan and China, PSK and PSP were well tolerated without significant side effects 667778798081. They also prolonged the survival of some patients with carcinoma and non-lymphoid leukemia.

Ganoderma polysaccharides are β-glucans derived from Ganoderma lucidum (Lingzhi, Reishi). While β-glucan is the major component of the Ganoderma mycelium, it is only a minor component in the Ganoderma spore 7. The main active ingredient in the Ganoderma spore extract is triterpenoid which is cytotoxic in nature. In an open-label study on patients with advanced lung cancer, thirty-six patients were treated with 5.4 g/day Ganoderma polysaccharides for 12 weeks with inconclusive variable and results on the cytokines profiles 82. Another study on 47 patients with advanced colorectal cancer using the same dosage and period again demonstrated similar variable immune response patterns 83. These results highlight the inconsistency of clinical outcomes in using immune enhancing herbal extracts clinically, which may partly be due to the impurity of the products used.