Improvement of interferon-gamma sialylation in Chinese hamster ovary cell culture by feeding of N-acetylmannosamine.

Because the presence of sialic acid can extend circulatory lifetime, a high degree of sialylation is often a desirable feature of therapeutic glycoproteins. In this study, the incomplete intracellular sialylation of interferon-gamma (IFN-gamma), produced by Chinese hamster ovary cell culture, was minimized by supplementing the culture medium with N-acetylmannosamine (ManNAc), a direct intracellular precursor for sialic acid synthesis. By introducing 20 mM ManNAc into the culture medium, incompletely sialylated biantennary glycan structures were reduced from 35% to 20% at the Asn97 glycosylation site. This effect was achieved without affecting cell growth or product yield. The intracellular pool of CMP-sialic acid, the nucleotide sugar substrate for sialyltransferase, was also extracted and quantified by HPLC. Feeding of 20 mM ManNAc increased this intracellular pool of CMP-sialic acid by nearly thirtyfold compared with unsupplemented medium. When radiolabeled ManNAc was used to trace the incorporation of the precursor, it was found that supplemental ManNAc was exclusively incorporated into IFN-gamma as sialic acid and that, at 20 mM ManNAc feeding, nearly 100% of product sialylation originated from the supplemental precursor.

Cytokines in nasal fluids from school children with seasonal allergic rhinitis.

Allergic rhinitis is a particularly good model for studies of cytokine production in vivo. In this study the occurrence of the cytokines IL-4, IL-5, IL-10 and IFN-gamma as well as the soluble receptor for IL-4 in nasal lavage fluids were assayed in 38 school children, with seasonal allergic rhinitis, and 19 healthy age-matched, non-atopic controls, using highly sensitive enzyme immunoassays. IL-4 levels in patients with seasonal allergic rhinitis were markedly increased in comparison with those in non-atopic controls or in atopic patients before the start of the pollen season. In controls, but not in the atopic patients, levels of IFN-gamma and IL-5 were significantly higher in specimens obtained during the pollen season than in those obtained outside the season. The IL-4/IFN-gamma ratios were significantly higher in atopic than in non-atopic subjects and further increased in atopic patients during the season. In addition to IL-4, elevated levels of IL-10 were observed in association with seasonal rhinitis. Following treatment with a topical steroid (budesonide) there was a statistically significant increase of the levels of soluble IL-4 receptor. These findings indicate that nonatopic and atopic individuals react to pollen exposure with distinct cytokine patterns in agreement with the Th1/Th2 concept. Topical steroids may possibly decrease inflammation by increasing the formation of soluble IL-4 receptor.

Glycoform heterogeneity of porcine interferon-gamma expressed in Sf9 cells.

Porcine interferon-gamma (SfPoIFN-gamma) was expressed with high efficiency in Spodoptera frugiperda (Sf9) cells by means of the baculovirus expression system. Up to 10(5) U/ml of antivirally active SfPoIFN-gamma could be tracked down in the culture medium at 64 h postinfection. Three proteins (17, 19, and 21 kDa), which under nondenaturing conditions primarily exist as mutual-dimeric combinations, were purified by immunoaffinity chromatography. Carbohydrate labeling and kinetic deglycosylation studies suggested that the 19- and 21-kDa proteins are N-glycosylated variants of a single 17-kDa protein carrying no N-linked sugars, in which one respectively two N-glycosylation sequons are occupied by glycans of 2 kDa. Both the quantitative recovery of SfPoIFN-gamma from a Con A column at 0.2 M methyl-alpha-mannopyranoside and the results of lectin blots, revealing strong affinity of the 19- and 21-kDa species for Galanthus nivalis agglutinin, support the presence of N-glycosidically linked high mannose-type chains in the carbohydrate moiety of SfPoIFN-gamma. Intriguingly, both 19- and 21-kDa glycoforms, but not their sialidase-treated derivatives, showed clear reactivity with the Sambucus nigra and Maackia amurensis agglutinins. These agglutinins specifically recognize sialic acid linked alpha(2-6) and alpha(2-3), respectively, to penultimate galactose residues. Their affinity for the larger glycoforms of PoIFN-gamma suggests that the biosynthetic pathways in Sf9 cells are able to modify oligomannose structures to complex or hybrid glycans.