Chemistry and biology of oligovalent ß-(1→2)-linked oligomannosides: new insights into carbohydrate-based adjuvants in immunotherapy.

A series of oligovalent carbohydrate assemblies (ranging from mono- to pentavalent), derived from three structurally different ß-linked or ß-(1→2)-linked mannosides, has been chemically synthesized, and the respective compounds have been biologically evaluated in order to investigate their immunostimulatory properties. The Crich methodology for ß-mannosylation was successfully utilized to introduce the ß-linkages, and a click chemistry protocol was utilized to generate the oligovalent derivatives. A convenient protecting group strategy involving the simultaneous use of both p-methoxybenzyl and benzylidene groups was employed, which allowed a simple and cost-effective global deprotection step. The immunomodulatory properties of the synthesized multivalent mannosides were evaluated by assessing cytokine production in human white blood cell cultures. The Th2-type cytokines interleukin-4 and interleukin-5 (IL-4 and IL-5), the Th1 cytokine interferon-γ (IFN-γ), the Treg cytokine IL-10, and the pro-inflammatory cytokine tumor necrosis factor (TNF) were included in the screening. A single trivalent acetylated mannobiose derivative was identified as a potent inducer of Treg and Th1 immune response, resulting in strong IL-10 and moderate IFN-γ productions dose-dependently, while inducing no Th2 cytokine response. The immunomodulatory properties of this trivalent mannoside were further studied in vitro in allergen (Bet v)-stimulated human peripheral blood mononuclear cell cultures of birch pollen allergic subjects. Stimulation with birch pollen induced strong IL-4 and IL-5 responses, which could be suppressed by the trivalent acetylated mannobiose derivative. The IL-10 response was also suppressed, whereas the production of IFN-γ was strongly enhanced. The results suggest that the identified lead compound has suppressive effects on the Th2-type allergic inflammatory response and shows potential as a possible lead adjuvant for the specific immunotherapy of allergies.

Treatment of chronic hepadnavirus infection in a woodchuck animal model with an inhibitor of protein folding and trafficking.

A novel strategy for anti-viral intervention of hepatitis B virus (HBV) through the disruption of the proper folding and transport of the hepadnavirus glycoproteins is described. Laboratory reared woodchucks chronically infected with woodchuck hepatitis virus (WHV) were treated with N-nonyl-deoxynojirimycin (N-nonyl-DNJ), an inhibitor of the endoplasmic reticulum (ER) alpha-glucosidases. The woodchucks experienced significant dose dependent decreases in enveloped WHV, resulting in undetectable amounts in some cases. The reduction in viremia correlated with the levels of hyperglucosylated glycan in the serum of treated animals. This correlation supports the mechanism of action associated with the drug and highlights the extreme sensitivity of the virus to this type of glycan inhibitor. At N-nonyl-DNJ concentrations that prevented WHV secretion, the glycosylation of most serum glycoproteins appeared unaffected, suggesting great selectivity for this class of therapeutics. Indeed, this may account for the low toxicity of the compound over the treatment period. We provide the first evidence that glucosidase inhibitors can be used in vivo to alter specific steps in the N-linked glycosylation pathway and that this inhibition has anti-viral effects.

Serum beta-(1----4)-galactosyltransferase activity with synthetic low molecular weight acceptor in human ovarian cancer.

A modified procedure was developed for the determination of UDP-galactose: 2-acetamido-2-deoxy-glucopyranoside beta-(1----4)-galactosyltransferase (GT) in human serum which employed the synthetic substrates p-nitrophenyl 6-0-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-D-galactopyranoside and p-nitrophenyl 6-0-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D- mannopyranoside as acceptors. The enzyme products were identified by thin layer chromatography with authentic reference compounds, and the galactosyl linkage was characterized by hydrolysis with beta-D-galactosidase from jack beans. The diagnostic value of this GT for ovarian cancer was tested by measuring the serum enzyme activity in 28 ovarian cancer patients with disease, 20 ovarian cancer patients with no clinical evidence of disease, and 22 healthy females. Although the level of the enzyme activity was significantly higher (P less than 0.002) in the serum of patients with active disease when compared to healthy controls, an appreciable overlap of enzyme activity was found between them. Also, no correlation was found between enzyme activity and tumor size. Differences in methodology and selection of patients makes it difficult to compare results from other reports. However, based on our improved assay procedure, we suggest caution should be exercised in evaluating the merits of GT as a diagnostic marker for ovarian cancer.