Evaluation of innate immune stimulating activity of polysaccharides using a silkworm (Bombyx mori) muscle contraction assay

Abstract

In silkworm larvae, the mature form of paralytic peptide (PP), an insect cytokine, is produced from pro-PP in association with activation of innate immune responses, resulting in slow muscle contraction. We utilized this reaction, muscle contraction in silkworms coupled with innate immunity stimulation, to quantitatively measure the innate immune stimulating activity of various natural polysaccharides. β-Glucan of Gyrophora esculenta (GE-3), fucoidan from sporophyll of Undaria pinnatifida, and curldan induced silkworm muscle contraction. We further demonstrated that GE-3 had therapeutic effects on silkworms infected by baculovirus. Based on these findings, we propose that the silkworm muscle contraction assay is useful for screening substances that stimulate innate immunity before evaluating therapeutic effectiveness in mammals.

Lactic acid bacteria activating innate immunity improve survival in bacterial infection model of silkworm

“…Our group found an active cytokine, paralytic peptide (PP) that induces muscle contraction (3). We have screened innate immunity activating substances by using silkworm muscle specimens (4,5). This screening method has the following advantages compared to conventional screening using mammalian innate immune cells like macrophages.…”

Structural analysis of an innate immunostimulant from broccoli, <i>Brassica oleracea</i> var. <i>italica </i>

“…We established an assay using the silkworm based on muscle contraction, which is associated with activation of innate immunity (6), and purified a polysaccharide with innate immunestimulating activity from green tea extracts (7). We also evaluated the activities of polysaccharides from natural origins using the silkworm muscle contraction assay (8). The silkworm muscle contraction assay has several advantages for evaluating innate immunostimulants (6)(7)(8).…”

“…We also evaluated the activities of polysaccharides from natural origins using the silkworm muscle contraction assay (8). The silkworm muscle contraction assay has several advantages for evaluating innate immunostimulants (6)(7)(8). The system does not respond to lipopolysaccharide (LPS) due to presence of LPS-absorbing proteins in the hemolymph, and thus potential LPS contamination of the assay is negligible.…”

Characterization of the chemical structure and innate immune-stimulating activity of an extracellular polysaccharide from <i>Rhizobium</i> sp. strain M2 screened using a silkworm muscle contraction assay

“…Although we could not determine the binding position of the succinate in the M2 EPS, succinate groups bind to glucose residues in the side-chains of succinoglycan from various microbial sources (24,25). The glycan structure of the side-chain of deacylated and depyruvylated M2 EPS is a β-1,3/6-glucan with the following structure: β-Glcp-(1→3)-β-Glcp-(1→3)-β-Glcp-(1→6)-β-Glcp-(1→6)-β-Glcp-(1→ Previously, the innate immunostimulating activities of β-1,3/6-glucan from various sources were evaluated using the silkworm muscle contraction assay (9), and the activities varied according to the origin. Unmasking of the β-1,3/6-glucan structure of the side chain by deacylation and depyruvylation may enhance the innate immune-stimulating activity of M2 EPS.…”

“…We established an assay using the silkworm based on muscle contraction, which is associated with the activation of innate immunity (7), and isolated a polysaccharide with innate immune-stimulating activity from green tea (8). We also evaluated the activities of natural polysaccharides from various origins using the silkworm muscle contraction assay (9). This assay system does not respond to lipopolysaccharides (LPS) due to the presence of LPS-absorbing proteins in the silkworm hemolymph, which may be highly advantageous for screening innate immune-stimulating compounds from Gram negative bacteria, as contamination of the assay system by LPS during the first screening is negligible.…”