Intravenous immunoglobulin G (IVIg) is widely used against a range of clinical symptoms. For its use in immune modulating therapies such as treatment of immune thrombocytopenic purpura high doses of IVIg are required. It has been suggested that only a fraction of IVIg causes this anti immune modulating effect. Recent studies indicated that this fraction is the Fc-sialylated IgG fraction. The aim of our study was to determine the efficacy of IVIg enriched for sialylated IgG (IVIg-SA (+)) in a murine model of passive immune thrombocytopenia (PIT). We enriched IVIg for sialylated IgG by Sambucus nigra agglutinin (SNA) lectin fractionation and determined the degree of sialylation. Analysis of IVIg-SA (+) using a lectin-based ELISA revealed that we enriched predominantly for Fab-sialylated IgG, whereas we did not find an increase in Fc-sialylated IgG. Mass spectrometric analysis confirmed that Fc sialylation did not change after SNA lectin fractionation. The efficacy of sialylated IgG was measured by administering IVIg or IVIg-SA (+) 24 hours prior to an injection of a rat anti-mouse platelet mAb. We found an 85% decrease in platelet count after injection of an anti-platelet mAb, which was reduced to a 70% decrease by injecting IVIg (p<0.01). In contrast, IVIg-SA (+) had no effect on the platelet count. Serum levels of IVIg and IVIg-SA (+) were similar, ruling out enhanced IgG clearance as a possible explanation. Our results indicate that SNA lectin fractionation is not a suitable method to enrich IVIg for Fc-sialylated IgG. The use of IVIg enriched for Fab-sialylated IgG abolishes the efficacy of IVIg in the murine PIT model.

Previously, we observed in a rat model that intravenous administration of intramuscular immunoglobulin preparations induced a long-lasting hypotension, which appeared to be associated with the presence of IgG polymers and dimers in the preparations, but unrelated to complement activation. We found evidence that this hypotensive response is mediated by platelet-activating factor (PAF) produced by macrophages. In this study, we compared the vasoactive effects of 16 intravenous immunoglobulin (IVIG) products from 10 different manufacturers, in anesthetized rats. Eight of the IVIG preparations showed no hypotensive effects (less than 15% decrease), whereas the other 8 had relatively strong effects (15%-50% decrease). The hypotensive effects correlated with the IgG dimer content of the preparations. Pretreatment of the rats with recombinant PAF acetylhydrolase completely prevented the hypotensive reaction on IVIG infusion, and administration after the onset of hypotension resulted in normalization of the blood pressure. We also observed PAF production on in vitro incubation of human neutrophils with IVIG, which could be blocked by anti-Fcγ receptor antibodies. This indicates that induction of PAF generation may also occur in a human system. Our findings support the hypothesis that the clinical side effects of IVIG in patients may be caused by macrophage and neutrophil activation through interaction of IgG dimers with Fcγ receptors. Because phagocyte activation may also lead to the release of other inflammatory mediators, recombinant PAF acetylhydrolase (rPAF-AH) provides a useful tool to determine whether PAF plays a role in the clinical side effects of IVIG. If so, rPAF-AH can be used for the treatment of those adverse reactions.

The manufacturing process of Nanogam comprises two effective steps for the reduction of LE viruses and one for NLE viruses. In addition, the precipitation of Cohn fraction III and the presence of neutralizing antibodies contribute to the total virus-reducing capacity of Nanogam. The overall virus-reducing capacity was > 15 log(10) for LE viruses. For the NLE viruses B19, CPV and EMC, the overall virus-reducing capacities were > 10, > 7 and > 9 log(10), respectively. Including the contribution of immune neutralization, the overall virus-reducing capacity for B19 and HAV is estimated to be > 10 log(10).

Novikoff ascites tumor cells contain a UDP‐GlcNAc:β‐galactoside β1→6‐N‐acetylglucosaminyltransferase (β6‐GlcNAc‐transferase B) that acts on galactosides and N‐acetylgalactosaminides in which the accepting sugar is β1→3 substituted by a Gal or GlcNAc residue. Characterization of enzyme products by 1H‐NMR and methylation analysis indicates that an Rβ1→3(GlcNAcβ1→6)Gal‐ branching point is formed such as occurs in blood‐group‐I‐active substances. The enzyme does not show an absolute divalent cation requirement and 20 mM EDTA is not inhibitory. The activity is strongly inhibited by Triton X‐100 at concentrations of ≥ 0.2%. Competition studies suggest that a single enzyme acts on Galβ1→3Galβ1→4Glc, GlcNAcβ1→3Galβ1→4GlcNAc and GlcNAcβ1→3GalNAcα‐O‐benzyl (Km values 0.71, 0.83 and 0.53 mM, respectively). Galβ→3Galβ1→4Glc as an acceptor substrate for β6‐GlcNAc‐transferase B does not inhibit the incorporation of GlcNAc in β1→6 linkage to the terminal Gal residues of asialo‐α1‐acid glycoprotein catalyzed by a β‐galactoside β1→6‐N‐acetylglucosaminyltransferase (β6‐GlcNAc‐transferase A) previously described in Novikoff ascites tumor cells [D. H. Van den Eijnden, H. Winterwerp, P. Smeeman & W. E. C. M. Schiphorst (1983) J. Biol. Chem. 258, 3435–3437]. Neither is Triton X‐100 at a concentration of 0.8% inhibitory for the activity of β6‐GlcNAc‐transferase A. This activity is absent from hog gastric mucosa microsomes, which has been described to contain high levels of β6‐GlcNAc‐transferase B. [F. Piller, J. P. Cartron, A. Maranduba, A. Veyrières, Y. Leroy & B. Fournet (1984) J. Biol. Chem. 259, 13385–13390]. Our results show that Novikoff tumor cells contain two β‐galactoside β‐galactoside β6‐GlcNAc‐transferases, which differ in acceptor specificity and tolerance towards Triton X‐100. A role for these enzymes in the synthesis of branched polylactosaminoglycans and of O‐linked oligosaccharide core structures having blood‐group I activity is proposed.

No abstract