Abstract: OBJECTIVE:: Besides its role in regulation of the complement and contact system, C1-esterase inhibitor has other immunomodulating effects that could prove beneficial in patients with acute inflammation such as during sepsis or after trauma. We examined the immunomodulating properties of C1-esterase inhibitor during human experimental endotoxemia, in which the innate immune system is activated in the absence of activation of the classic complement pathway. DESIGN:: Double-blind placebo-controlled study. SETTING:: Research intensive care unit of the Radboud University Nijmegen Medical Centre. SUBJECTS:: Twenty healthy volunteers. INTERVENTIONS:: Intravenous injection of 2 ng/kg Escherichia coli lipopolysaccharide. Thirty minutes thereafter (to prevent binding of lipopolysaccharide), C1-esterase inhibitor concentrate (100 U/kg, n = 10) or placebo (n = 10) was infused. MEASUREMENTS AND MAIN RESULTS:: Pro- and anti-inflammatory mediators, markers of endothelial and complement activation, hemodynamics, body temperature, and symptoms were measured. C1-esterase inhibitor reduced the release of proinflammatory cytokines as well as C-reactive protein (peak levels of: interleukin-6 1521 +/- 209 vs. 932 +/- 174 pg/mL [p = .04], tumor necrosis factor-alpha 1213 +/- 187 vs. 827 +/- 167 pg/mL [p = .10], monocyte chemotactic protein-1 6161 +/- 1302 vs. 3373 +/- 228 pg/mL [p = .03], interleukin-1beta 34 +/- 5 vs. 23 +/- 2 pg/mL [p < .01], C-reactive protein 39 +/- 4 vs. 29 +/- 2 mg/L [p = .02]). In contrast, release of the anti-inflammatory cytokine interleukin-10 was increased by C1-esterase inhibitor (peak level 73 +/- 11 vs. 121 +/- 18 pg/mL, p = .02). The increase in interleukin-1 receptor antagonist tended to be smaller in the C1-esterase inhibitor group, but this effect did not reach statistical significance (p = .07). Markers for endothelial activation were increased after lipopolysaccharide infusion, but no significant differences between groups were observed. The lipopolysaccharide-induced changes in heart rate, blood pressure, body temperature, and symptoms (all p < .001 over time) were not influenced by C1-esterase inhibitor. Complement fragment C4 was not increased after lipopolysaccharide challenge. CONCLUSIONS:: This study is the first to demonstrate that C1-esterase inhibitor exerts anti-inflammatory effects in the absence of classic complement activation in humans.
Abstract: We studied the efficacy of virus reduction by three process steps (polyethylene glycol 4000 (PEG) precipitation, pasteurization, and 15nm virus filtration) in the manufacturing of C1-inhibitor NF. The potential prion removing capacity in this process was estimated based on data from the literature. Virus studies were performed using hepatitis A virus (HAV) and human immunodeficiency virus (HIV) as relevant viruses and bovine viral diarrhea virus (BVDV), canine parvovirus (CPV) and pseudorabies virus (PRV) as model viruses, respectively. In the PEG precipitation step, an average reduction in infectious titer of 4.5log(10) was obtained for all five viruses tested. Pasteurization resulted in reduction of infectious virus of >6log(10) for BVDV, HIV, and PRV; for HAV the reduction factor was limited to 2.8log(10) and for CPV it was zero. Virus filtration (15nm) reduced the infectious titer of all viruses by more than 4.5log(10). The overall virus reducing capacity was >16log(10) for the LE viruses. For the NLE viruses CPV and HAV, the overall virus reducing capacities were >8.7 and >10.5log(10), respectively. Based on literature and theoretical assumptions, the prion reducing capacity of the C1-inhibitor NF process was estimated to be >9log(10).
Abstract: BACKGROUND AND OBJECTIVES: Producers of plasma derivatives continuously improve the viral safety of their products by, for example, introducing additional virus-reducing steps into the manufacturing process. Here we present virus-elimination studies undertaken for a number of steps employed in a new manufacturing process for liquid intravenous immunoglobulin (Nanogam) that comprises two specific virus-reducing steps: a 15-nm filtration step combined with pepsin treatment at pH 4.4 (pH 4.4/15NF); and solvent-detergent (SD) treatment. The manufacturing process also includes precipitation of Cohn fraction III and viral neutralization, which contribute to the total virus-reducing capacity of the manufacturing process. In addition, the mechanism and robustness of the virus-reducing steps were studied. MATERIALS AND METHODS: Selected process steps were studied with spiking experiments using a range of lipid enveloped (LE) and non-lipid-enveloped (NLE) viruses. The LE viruses used were bovine viral diarrhoea virus (BVDV), human immunodeficiency virus (HIV) and pseudorabies virus (PRV); the NLE viruses used were parvovirus B19 (B19), canine parvovirus (CPV) and encephalomyocarditis virus (EMC). After spiking, samples were collected and tested for residual infectivity, and the reduction factors were calculated. For B19, however, removal of B19 DNA was measured, not residual infectivity. To reveal the contribution of viral neutralization, bovine parvovirus (BPV) and hepatitis A virus (HAV) were used. RESULTS: For the pH 4.4/15NF step, complete reduction (> 6 log(10)) was demonstrated for all viruses, including B19, but not for CPV (> 3.4 but < or = 4.2 log(10)). Robustness studies of the pH 4.4/15NF step with CPV showed that pH was the dominant process parameter. SD treatment for 10 min resulted in complete inactivation (> 6 log(10)) of all LE viruses tested. Precipitation of Cohn fraction III resulted in the significant removal (3-4 log(10)) of both LE and NLE viruses. Virus-neutralization assays of final product revealed significant reduction (> or = 3 log(10)) of both BPV and HAV. CONCLUSIONS: 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).
Abstract: BACKGROUND: Intravenous immunoglobulin (IVIG) products formulated as a liquid instead of a powder have become commercially available. Preferably, such liquid products should not alter after storage outside the refrigerator. Therefore, a thorough characterization of immunoglobulin G (IgG) fragmentation at various storage temperatures is required. STUDY DESIGN AND METHODS: Storage experiments with liquid IVIG products from five manufacturers were performed at 4, 25, and 37 degrees C and IgG fragments were analyzed. RESULTS: Storage of liquid IVIG products at 4 degrees C resulted in negligible alterations, whereas an increase of IgG fragments was observed after prolonged storage at elevated temperatures. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis of five liquid IVIG products revealed three IgG fragments (12, 26, and 54 kDa) in all products. Fragments of similar molecular mass were produced upon incubations of IgG with blood-derived proteases. N-terminal amino acid sequencing revealed the cleavage site of these fragments, suggesting human neutrophil elastase to cause the 12-kDa fragment. The presence of elastase in liquid IVIG was confirmed by enzyme-linked immunosorbent assay. The origin of the 26- and 54-kDa fragments, both with an aspartic acid residue at the cleavage site, could not be determined unambiguously. CONCLUSION: IgG fragmentation in liquid IVIG is negligible when stored in the refrigerator. Only after prolonged storage at elevated temperature does proteolytic degradation of IgG become apparent.
Abstract: 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-Fcgamma 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 Fcgamma 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. (Blood. 2000;95:1856-1861)
Abstract: A specific and fast method for the determination of N-acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three transferase products formed from the common acceptor oligosaccharide substrate GlcNAc beta 1----2Man alpha 1----3(GlcNAc beta 1----2Man alpha 1---- 6)Man beta 1----4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breakdown of the donor substrate UDP-[14C]GlcNAc. Using this assay system N-acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.
Abstract: An N-acetylglucosaminyltransferase has been partially purified from Novikoff tumor cell ascites fluid by affinity chromatography on concanavalin A-Sepharose. The enzyme was obtained in a highly concentrated form after lyophilization. The enzyme appeared to be highly specific for acceptor oligosaccharides and glycoproteins carrying a terminal Gal beta 1----4GlcNAc beta 1----R unit. Characterization of products formed by the enzyme in vitro by methylation analysis and 1H NMR spectroscopy revealed that the enzyme catalyzed the formation of a GlcNAc beta 1----3Gal beta 1----4GlcNAc beta-R sequence. The enzyme therefore could be described as an UDP-GlcNAc:Gal beta 1----4GlcNAc beta-R beta 1----3-N-acetylglucosaminyltransferase. Acceptor specificity studies with oligosaccharides that form part of N-glycans revealed that the presence of a Gal beta 1----4GlcNAc beta 1----2(Gal beta 1----4GlcNAc beta 1----6)Man pentasaccharide in the acceptor structure is a requirement for optimal activity. Studies on the branch specificity of the enzyme showed that the branches of this pentasaccharide structure, when contained in tri- and tetraantennary oligosaccharides, are highly preferred over other branches for attachment of the 1st and 2nd mol of GlcNAc into the acceptor molecule. The enzyme also showed activity toward oligosaccharides related to blood group I- and i-active polylactosaminoglycans. In addition the enzyme together with calf thymus UDP-Gal:GlcNAc beta-R beta 1----4-galactosyltransferase was capable of catalyzing the synthesis of a series of oligomers of N-acetyllactosamine. Competition studies revealed that all acceptors were acted upon by a single enzyme species. It is concluded that the N-acetylglucosaminyltransferase functions in both the initiation and the elongation of polylactosaminoglycan chains of N-glycoproteins and possibly other glycoconjugates.
Abstract: By use of a new assay method based on HPLC, GlcNAc-transferase III, IV and V activities were determined in HL-60 cells, differentiated HL-60 cells and normal myeloid cells. Differentiation along the monocytic lineage with 1 alpha,25-dihydroxyvitamin D3 resulted in increased GlcNAc-transferase IV and decreased GlcNAc-transferase III activity. Differentiation along the myeloid lineage with retinoic acid resulted in a decrease in GlcNAc-transferase III activity. Although differentiated HL-60 cells show a changed GlcNAc-transferase pattern, they do not resemble normal myeloid cells. Macrophages and granulocytes are characterized by a very low level of GlcNAc-transferase III activity whereas differentiated HL-60 cells still contain this activity. This is the first demonstration of GlcNAc-transferase IV and V activity in a human cell.
Abstract: Novikoff ascites tumor cells contain a UDP-GlcNAc:beta-galactoside beta 1----6-N-acetylglucosaminyltransferase (beta 6-GlcNAc-transferase B) that acts on galactosides and N-acetylgalactosaminides in which the accepting sugar is beta 1----3 substituted by a Gal or GlcNAc residue. Characterization of enzyme products by 1H-NMR and methylation analysis indicates that an R beta 1----3(GlcNAc beta 1----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 greater than or equal to 0.2%. Competition studies suggest that a single enzyme acts on Gal beta 1----3Gal beta 1----4Glc, GlcNAc beta 1----3Gal beta 1----4GlcNAc and GlcNAc beta 1----3GalNAc alpha-O-benzyl (Km values 0.71, 0.83 and 0.53 mM, respectively). Gal beta----3Gal beta 1----4Glc as an acceptor substrate for beta 6-GlcNAc-transferase B does not inhibit the incorporation of GlcNAc in beta 1----6 linkage to the terminal Gal residues of asialo-alpha 1-acid glycoprotein catalyzed by a beta-galactoside beta 1----6-N-acetylglucosaminyltransferase (beta 6-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 beta 6-GlcNAc-transferase A. This activity is absent from hog gastric mucosa microsomes, which has been described to contain high levels of beta 6-GlcNAc-transferase B. [F. Piller, J. P. Cartron, A. Maranduba, A. Veyrières, Y. Leroy & B. Fournet (1984) J. Biol. Chem. 259, 13,385-13,390]. Our results show that Novikoff tumor cells contain two beta-galactoside beta 6-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.
Abstract: A sensitive HPLC method for the assay of UDP-GlcNAc:beta-galactoside beta 1----3-N-acetylglucosaminyltransferase activity was developed. Using lactose as an acceptor, the formation of the product GlcNAc beta 1----3Gal beta 1----4Glc can be determined without interference by substrates resulting from enzymatic and chemical breakdown of the donor substrate UDP-GlcNAc. The method is very specific since products of other transferase reactions, which potentially may be formed in the incubations in vitro, elute at positions different from that of GlcNAc beta 1----3Gal beta 1----4Glc. By use of this assay method it could be demonstrated that normal and malignant hematopoietic cells and cell-lines, with the exception of erythrocytes and reticulocytes, contain beta 1----3-N-acetylglucosaminyltransferase activity.
Abstract: A UDP-GlcNAc:R1-beta 1-3Gal(NAc)-R2 [GlcNAc to Gal(NAc)] beta 6-N-acetylglucosaminyltransferase activity from pig gastric mucosa microsomes catalyzes the formation of GlcNAc beta 1-3(GlcNAc beta 1-6)Gal-R from GlcNAc beta 1-3Gal-R where -R is -beta 1-3GalNAc-alpha-benzyl or -beta 1-3(GlcNAc beta 1-6)GalNAc-alpha-benzyl. This enzyme is therefore involved in the synthesis of the I antigenic determinant in mucin-type oligosaccharides. The enzyme also converts Gal beta 1-3Gal beta 1-4Glc to Gal beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc. The enzyme was stimulated by Triton X-100 at concentrations between 0 and 0.2% and was inhibited by Triton X-100 at 0.5%. There is no requirement for Mn2+ and the enzyme activity is reduced to 65% in the presence of 10 mM EDTA. Enzyme products were purified and identified by proton NMR, methylation analysis and beta-galactosidase digestion. Competition studies suggest that this pig gastric mucosal beta 6-GlcNAc-transferase activity is due to the same enzyme that converts Gal beta 1-3GalNAc-R to mucin core 2, Gal beta 1-3(GlcNAc beta 1-6)GalNAc-R, and GlcNAc beta 1-3GalNAc-R to mucin core 4, GlcNAc beta 1-3(GlcNAc beta 1-6)GalNAc-R. Substrate specificity studies indicate that the enzyme attaches GlcNAc to either Gal or GalNAc in beta (1-6) linkage, provided these residues are substituted in beta (1-3) linkage by either GlcNAc or Gal. The insertion of a GlcNAc beta 1-3 residue into Gal beta 1-3GalNAc-R to form GlcNAc beta 1-3Gal beta 1-3GalNAc-R prevents insertion of GlcNAc into GalNAc. These studies establish several novel pathways in mucin-type oligosaccharide biosynthesis.
Abstract: Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubilized by treatment of the mitochondria with glucose 6-phosphate and partly purified. The solubilized enzyme was compared with the cytosolic enzyme fraction. The solubilized and cytosolic enzymes were also compared with the enzyme bound to the mitochondrial membrane. The following observations were made. 1. There is no difference in electrophoretic mobility on cellulose-acetate between the cytosolic and the solubilized enzyme. Both fractions are hexokinase isoenzyme I. 2. There is no difference in kinetic parameters between the cytosolic or solubilized enzymes (P less than 0.001). For the cytosolic enzyme Km for glucose was 0.067 mM (S.E. = 0.024, n = 7); Km for MgATP2- was 0.42 mM (S.E. = 0.13, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.084 mM (S.E. = 0.011, n = 5). For the solubilized enzyme Km for glucose was 0.071 mM (S.E. = 0.021, n = 6); Km for MgATP2- was 0.38 mM (S.E. = 0.11, n = 6) and Ki,app for glucose 1,6-diphosphate was 0.074 mM (S.E. = 0.010, n = 5). However when bound to the mitochondrial membrane, the enzyme has higher affinities for its substrates and a lower affinity for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction Km for glucose was 0.045 mM (S.E. = 0.013, n = 7); Km for MgATP2- was 0.13 mM (S.E. = 0.02, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.33 mM (S.E. = 0.03, n = 5). 3. The cytosolic and solubilized enzyme could be (re)-bound to depleted mitochondria to the same extent and with the same affinity. Limited proteolysis fully destroyed the enzyme's ability to bind to depleted mitochondria. 4. Our data support the hypothesis that soluble- and solubilizable enzyme from rat brain are one and the same enzyme, and that there is a simple equilibrium between the enzyme in these two pools.
Abstract: 1. The effects of oestradiol-17 beta on processes of synthesis in the pyloric caeca of female Asterias rubens were studied. 2. In vitro treatment with 2.5 X 10(-7) M oestradiol-17 beta resulted in significantly higher RNA levels. 3. In vivo treatment with oestradiol-17 beta resulted in higher lipid levels in the pyloric caeca, but RNA levels, protein levels and the incorporation of 6-[14C]orotic acid, L-leucine-[14C] and sodium 1-[14C] acetate into RNA, proteins and lipids, respectively were not affected, neither were the indices of the gonads and pyloric caeca.
