**4.1. Biological pathways of activation**

The complement system is responsible for innate immunity and can be activated by either classical, alternative or lectin pathways. Using lentinan, pachyman and pachymaran polysaccharides, it has been demonstrated that 1,3-β-glucans use the alternative pathway of complement activation for generating an immune response since they show an increased consumption of C3 and C5 proteins from the complement cascade [57]. Additionally, opsonization is an important component of innate immune response. Typically complement protein C3b gets coated on pathogens and is later detected by complement receptor 1 or deactivated to form iC3b for regulation. In the presence of 1,3-β-glucans, iC3b can be detected by complement receptor 3 (CR3) or dectin-1 and this mechanism can be exploited for attacking cancer cells coated with iC3b [8]. Although the effect of CR3 has been negligible in immune activation in murine models [9,58], functional CR3 is essential for phagocytosis in human neutrophils [59].

Most of the existing studies have focused on macrophages and neutrophils but some 1,3-βglucans such as lentinan are responsible for stimulation of T-cells and natural killer cells and hence affect the acquired immune response [60,61]. Curdlan has also been used to demonstrate increased proliferation of lymphocytes, which can in turn enhance the immune response [62]. Oxidative burst is yet another immunomodulatory effect that has been demonstrated by 1,3-βglucans. While curdlan has shown the induction of inducible nitric oxide synthase in rat macrophages [63], other 1,3-β-glucans have shown an increase in the production of reactive oxygen species [62]. Some studies have demonstrated that immune activation is only possible by linear 1,3-β-glucans [64] while others emphasize that complex branching is important for most effective stimulation of immune response [65]. Although, several aspects of the biological pathways of activation have been discovered, further studies are necessary to gain a better understanding of the intricate interactions between 1,3-β-glucans and the immune system.

#### **4.2. Tumor suppression**

The use of 1,3-β-glucans in cancer therapy has been present in Japan since 1986, where they have been used for gastric, lung and cervical cancers [66]. Lentinan and pachymaran have demonstrated high tumor inhibition ratios of 99.6% and 96%, when tested against subcutaneous implantation of sarcoma 180 in mice [64]. It is hypothesized that the effect of lentinan and pachymaran is highly dependent on the activation of T-lymphocytes because the removal of the thymus from mice caused a suppression of antitumor effects from the 1,3-βglucans [60]. Additionally, it is also speculated that deactivation of protein helices might be important for antitumor effects because a study has demonstrated that only polysaccharides that deactivated bovine serum albumin showed antitumor activity [67]. Besides lentinan and pachymaran, other 1,3-β-glucans have also exemplified tumor suppression. Some of the prominent examples are scleroglucan with an inhibition ratio of 90.4%, curdlan with inhibition of 99-100% [68], grifolan with inhibition of 97.9% [69] and 1,3-β-glucan from *Agaricus blazei* with inhibition of 99.3% [65]. These results have been encouraging and hence 1,3-β-glucans have been used in combinatorial therapies with antibodies in implanted human tumor xenografts from melanoma, epidermoid carcinoma, breast carcinoma, metastatic lymphoma and daudi lymphoma. The mice had higher survival rates in the presence of 1,3-β-glucans as compared to treatment with antibodies alone [12].

Subsequently, water soluble 1,3-β-glucans have been derived to improve the usability of these polysaccharides. Some examples of these polysaccharides include carboxymethylpachymaran with tumor inhibition ratio of 99.6%, hydroxymethylpachymaran and hydroxypropylpachymaran with up to 100% tumor inhibition when assessed against solid sarcoma 180 at a dose of 5 mg/kg [70]. Several derivatives of curdlan including carboxymethyl, glucosyl, sulfoethyl and sulfopropyl attachments to the backbone have also retained antitumor activity [71,72]. These examples demonstrate the versatility of 1,3-β-glucans in cancer inhibition and thus these polysaccharides can be modified to suit the desired application.

### **4.3. Infection prevention**

564 The Complex World of Polysaccharides

resistance, disease immunity and wound healing [56].

**4.1. Biological pathways of activation** 

phagocytosis in human neutrophils [59].

**4.2. Tumor suppression** 

activation and this response has been utilized for varied applications including cancer

The complement system is responsible for innate immunity and can be activated by either classical, alternative or lectin pathways. Using lentinan, pachyman and pachymaran polysaccharides, it has been demonstrated that 1,3-β-glucans use the alternative pathway of complement activation for generating an immune response since they show an increased consumption of C3 and C5 proteins from the complement cascade [57]. Additionally, opsonization is an important component of innate immune response. Typically complement protein C3b gets coated on pathogens and is later detected by complement receptor 1 or deactivated to form iC3b for regulation. In the presence of 1,3-β-glucans, iC3b can be detected by complement receptor 3 (CR3) or dectin-1 and this mechanism can be exploited for attacking cancer cells coated with iC3b [8]. Although the effect of CR3 has been negligible in immune activation in murine models [9,58], functional CR3 is essential for

Most of the existing studies have focused on macrophages and neutrophils but some 1,3-βglucans such as lentinan are responsible for stimulation of T-cells and natural killer cells and hence affect the acquired immune response [60,61]. Curdlan has also been used to demonstrate increased proliferation of lymphocytes, which can in turn enhance the immune response [62]. Oxidative burst is yet another immunomodulatory effect that has been demonstrated by 1,3-βglucans. While curdlan has shown the induction of inducible nitric oxide synthase in rat macrophages [63], other 1,3-β-glucans have shown an increase in the production of reactive oxygen species [62]. Some studies have demonstrated that immune activation is only possible by linear 1,3-β-glucans [64] while others emphasize that complex branching is important for most effective stimulation of immune response [65]. Although, several aspects of the biological pathways of activation have been discovered, further studies are necessary to gain a better understanding of the intricate interactions between 1,3-β-glucans and the immune system.

The use of 1,3-β-glucans in cancer therapy has been present in Japan since 1986, where they have been used for gastric, lung and cervical cancers [66]. Lentinan and pachymaran have demonstrated high tumor inhibition ratios of 99.6% and 96%, when tested against subcutaneous implantation of sarcoma 180 in mice [64]. It is hypothesized that the effect of lentinan and pachymaran is highly dependent on the activation of T-lymphocytes because the removal of the thymus from mice caused a suppression of antitumor effects from the 1,3-βglucans [60]. Additionally, it is also speculated that deactivation of protein helices might be important for antitumor effects because a study has demonstrated that only polysaccharides that deactivated bovine serum albumin showed antitumor activity [67]. Besides lentinan and pachymaran, other 1,3-β-glucans have also exemplified tumor suppression. Some of the prominent examples are scleroglucan with an inhibition ratio of 90.4%, curdlan with inhibition Most common infections are caused by bacteria and fungi and since 1,3-β-glucans can induce inflammatory response, these glucans can be used for providing infection resistance. It has been shown that when administering 1,3-β-glucan to mice, their survival against *Staphylococcus aureus* infection increased from 70% to 97% [73]. Other 1,3-β-glucans such as glucan phosphate, laminarin and scleroglucan have been studied in detail for assessing their pharmacokinetic profile following oral administration in rats. It has been observed that these glucans are able to translocate from the gastrointestinal tract to systemic circulation. The glucans were able to increase secretion of interleukins, increase expression of dectin-1 on macrophages and increase expression of toll-like receptor 2 on dendritic cells. Thus, these effects increase the long-term survival of rats from 0% to 40% when challenged with *Candida albicans* fungal infection and from 0% to 50% when challenged with *Staphyloccous aureus* bacterial infection [74].

When considering larger mammals, infection resistance in pigs, dogs and horses has also found applications of 1,3-β-glucans. When piglets were fed with β-glucans after weaning, they demonstrated lower infection from enterotoxigenic *Escherichia coli*, which was highlighted by decreased diarrhoea and decreased content of inoculated *Escherichia coli* in the faeces as compared to control groups. These results present a significant advancement in veterinary medicine for pigs because the immunity of pigs usually suffers severely right after weaning and the current vaccines against *Escherichia coli* take a long period to be effective [20]. In the case of horses, the administration of 1,3-β-glucan to pregnant mares has been able to increase the cellular immune response in foals. This becomes very useful for preventing premature deaths of neonates [75]. While most studies have focused on the effects of 1,3-β-glucans on improving innate immunity, when these polysaccharides are administered to dogs, they showed enhancement in humoral immunity as indicated by changes in serum IgM and IgA levels [18].

1,3-β-glucans have also been used in aquatic animals as exemplified by the use of schizophyllan with 60-80% survival rate and lentinan and scleroglucan with 55-75% survival rate when tested against *Edwardsiella tarda* bacteria attack on *Cyrinus carpio L.* carp. The survival rate was also increased when infected with *Aeromonas hydrophila* with survival of 60% with schizophyllan, 70% with lentinan and 80% with scleroglucan administration. In the absence of glucans, the carp underwent complete mortality upon any infection [76]. These results have been repeatable when tilapia and grass carp is exposed to *Aeromonas hydrophila* [77]. As more studies are conducted on the use of 1,3-β-glucans, the quality of veterinary health care can be improved further and these results can eventually be transferred to human applications.

In addition to bacterial and fungal resistance, 1,3-β-glucans have also demonstrated promising results against malaria [78], herpes simplex virus [79] and human immunodeficiency virus (HIV) [80-82]. Expanding on HIV research, various complexes have been synthesized with curdlan sulfate in order to enhance the efficacy of the polysaccharide. Some prominent examples include covalent conjugation of azidothymidine to curdlan sulfate for drug delivery to the lymph nodes and bone marrow [83] and conjugation of fullerene C60 with curdlan for combining their anti-HIV effects [84].
