Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolia.

An alpha-D-glucan (RR1) composed of (1-->4) linked back bone and (1-->6) linked branches with a molecular mass of >550 kDa and exhibiting unique immune stimulating properties is isolated and characterized from the medicinal plant Tinospora cordifolia. This novel polysaccharide is noncytotoxic and nonproliferating to normal lymphocytes as well as tumor cell lines at 0-1000 microg/ml. It activated different subsets of the lymphocytes such as natural killer (NK) cells (331%), T cells (102%), and B cells (39%) at 100 microg/ml concentration. The significant activation of NK cells is associated with the dose-dependent killing of tumor cells by activated normal lymphocytes in a functional assay. Immune activation by RR1 in normal lymphocytes elicited the synthesis of interleukin (IL)-1beta (1080 pg/ml), IL-6 (21,833 pg/ml), IL-12 p70 (50.19 pg/ml), IL-12 p40 (918.23 pg/ml), IL-18 (27.47 pg/ml), IFN- gamma (90.16 pg/ml), tumor necrosis factor (TNF)-alpha (2225 pg/ml) and monocyte chemoattractant protein (MCP)-1 (2307 pg/ml) at 100 microg/ml concentration, while it did not induce the production of IL-2, IL-4, IL-10, interferon (IFN)-alpha and TNF-beta. The cytokine profile clearly demonstrates the Th1 pathway of T helper cell differentiation essential for cell mediated immunity, with a self-regulatory mechanism for the control of its overproduction. RR1 also activated the complements in the alternate pathway, demonstrated by a stepwise increase in C3a des Arg components. Incidentally, RR1 stimulation did not produce any oxidative stress or inducible nitric oxide synthase (iNOS) in the lymphocytes or any significant increase in nitric oxide production. The water solubility, high molecular mass, activation of lymphocytes especially NK cells, complement activation, Th1 pathway-associated cytokine profile, together with a low level of nitric oxide synthesis and absence of oxidative stress confer important immunoprotective potential to this novel alpha-D-glucan.

A monoclonal antibody reacts species-specifically with amylopectin granules of Eimeria bovis merozoites.

An IgG1 monoclonal antibody (mAb 35B9) developed against first-generation merozoites of Eimeria bovis was shown by immunoelectron microscopy to react selectively with antigens localized in amylopectin granules. Amylopectin does not contribute to the epitope, as enzymatic degradation of carbohydrates in the parasite did not alter the binding pattern of mAb 35B9. When tested by immunoblotting, despite its organelle specificity the mAb recognized a variety of E. bovis merozoite I components with predominant molecules of 135 and 200 kDa. The epitope was not affected by treatment with endoglycosidase H; thus, N-linked sugar residues should not be involved in it. Alkaline cleavage (beta-elimination), however, destroyed the epitope; thus, the involvement of O-linked carbohydrates cannot be excluded. Treatment of E. bovis merozoite extract with phospholipase C changed the binding pattern of mAb 35B9 in a way that suggests the presence of phosphorylcholine molecules on several antigens recognized by the mAb, albeit not belonging to the epitope but rather masking it. The epitope was not found in free sporozoites of E. bovis or young intracellular parasites up to day 4 after invasion of cells in vitro, whereas 5-day-old trophozoites were found to contain it. It seems to be species-specific, as it could not be shown in sporozoites or merozoites of E. tenella or in stages of several other Coccidia.