Wolfgang Teschner

Glycoproteomic characterization of butyrylcholinesterase from human plasma

Human butyrylcholinesterase (hBChE) is a highly glycosylated protein present in human plasma. The enzyme hydrolyses choline esters, for example benzoylcholine, butyrylthiocholine and acetylthiocholine as well as noncholine esters like heroin and aspirin. hBChE is primarily involved in neuronal transmission and is a potential bioscavenger of toxic organophosphates to protect acetylcholinesterase. A prerequisite for the therapeutic use of hBChE is a detailed characterization of this glycoprotein purified from human plasma. In this study, MS/MS could confirm most of the protein backbone, including the N‐ and the C‐terminus. Site‐specific analysis of all nine potential N‐glycosylation sites revealed mainly mono‐ and disialylated N‐glycans to be present on this glycoprotein. Sialic acids (Neu5Ac) are mainly α2,6‐linked, however a fraction of the N‐glycans contained Neu5Ac also in α2,3 linkage. On monosialylated N‐glycans, sialic acid is exclusively located on the 3‐arm and in α2,6 linkage, as verified by 2D‐HPLC and exoglycosidase digests of 2‐aminopyridine (PA)‐labelled N‐glycans. This first comprehensive glycoproteomic analysis of the important human plasma glycoprotein BChE did not give any indication of O‐glycosylation or any other kind of PTMs as previously postulated.

Immunomodulatory properties of human serum immunoglobulin A: anti-inflammatory and pro-inflammatory activities in human monocytes and peripheral blood mononuclear cells.

Our study investigated the immunomodulatory activities of human plasma‐derived serum immunoglobulin (Ig)A. Previous findings seem contradictory indicating either pro‐ or anti‐inflammatory activities. We used serum IgA purified from large plasma pools and studied the modulation of the release of cytokines and chemokines from resting and lipopolysaccharide (LPS, endotoxin)‐stimulated human adherent monocytes and human peripheral blood mononuclear cells (PBMC). Our results indicate that IgA down‐modulates the release of the pro‐inflammatory chemokines monocyte chemoattractant protein (MCP) 1, macrophage inflammatory protein (MIP) 1α and MIP1β from LPS‐stimulated PBMC and the release of MCP1, MIP1α and MIP1β from LPS‐stimulated monocytes. Furthermore, we confirmed previous reports that plasma‐derived serum IgA down‐modulates the release of the pro‐inflammatory cytokines, interleukin (IL)‐6 and tumour necrosis factor (TNF)‐α, from LPS‐stimulated monocytes and PBMC, and up‐regulates the release of IL‐1 receptor antagonist (IL‐1RA) from resting and LPS‐stimulated monocytes and resting PBMC. This IgA‐mediated up‐regulation of IL‐1RA is independent of the simultaneous up‐regulation of IL‐1β release, as shown by blocking the biological activity of IL‐1β with a neutralizing antibody. On the other hand, we also found an IgA‐induced pro‐inflammatory activity, namely IgA‐mediated up‐regutation of the release of pro‐inflammatory IL‐1β as well as down‐regulation of the anti‐inflammatory cytokines IL‐10 and IL‐12p40 from LPS‐stimulated monocytes and PBMC and a down‐regulation of transforming growth factor (TGF)‐β from resting and LPS‐stimulated PBMC. We conclude that human serum IgA has both an anti‐inflammatory and a pro‐inflammatory capacity and this dual capacity might contribute to the feedback mechanisms maintaining a balance between pro‐inflammatory and anti‐inflammatory activities.

A new liquid, intravenous immunoglobulin product (IGIV 10%) highly purified by a state-of-the-art process.

Background and Objectives  The ultimate goal was to generate an industrial‐scale process suitable to produce a high‐yield, safe and stable immunoglobulin G (IgG) preparation for intravenous administration, which is ready to use for customer convenience. This new liquid 10% IgG preparation (IGIV 10%) was compared to Gammagard SD, a licenced lyophilized immunoglobulin in biochemical and preclinical testing.

A new liquid intravenous immunoglobulin with three dedicated virus reduction steps: virus and prion reduction capacity

Background and Objectives  A new 10% liquid human intravenous immunoglobulin (US trade name: Gammagard Liquid; European trade name: KIOVIG) manufactured by a process with three dedicated pathogen inactivation/removal steps (solvent/detergent treatment, 35‐nm nanofiltration and low pH/elevated temperature incubation) was developed. The ability of the manufacturing process to inactivate/remove viruses and prions was investigated.

Removal of small nonenveloped viruses by antibody‐enhanced nanofiltration during the manufacture of plasma derivatives

BACKGROUND:  Filters with nominal pore sizes in the nanometer range are well‐established tools for enhancing the virus safety margins of plasma‐derived products, yet intrinsically less successful for smaller viruses such as hepatitis A virus (HAV) and human parvovirus B19 (B19V). The formation of virus‐antibody complexes increases the effective size of these smaller viruses and would thus improve their removal by nanofiltration. While the principle of virus removal by antibody‐dependent nanofiltration has been demonstrated with animal antisera and viruses spiked into human plasma product intermediates, the significance of these results remains unclear due to the potential contributions of xenoanti‐bodies and/or heteroagglutination in such heterologous systems.

Human IgG Subclasses: In Vitro Neutralization of and In Vivo Protection against West Nile Virus

ABSTRACT West Nile virus (WNV)-neutralizing intravenous immune globulins (IVIG) were fractionated into IgG subclasses, and the contribution of each subclass to in vitro neutralization of and in vivo protection against WNV was evaluated. The results indicate that IgG1 (i) is the main subclass induced following WNV infection of humans, (ii) contained nearly all the in vitro WNV neutralization capacity, and (iii) mediates effector functions in vivo that render it superior to other subclasses in protection against WNV. The importance of human IgG1 indicates that a candidate WNV vaccine should induce an immune response that includes WNV-specific IgG1.

Biochemical, molecular and preclinical characterization of a double-virus-reduced human butyrylcholinesterase preparation designed for clinical use.

Background and Objectives  A human plasma‐derived butyrylcholinesterase preparation manufactured on the industrial scale is described.

Efficacy and physiological effects of human butyrylcholinesterase as a post-exposure therapy against percutaneous poisoning by VX in the guinea-pig.

The physiological effects of human plasma-derived butyrylcholinesterase (huBuChE) administration and its modulation of the effects of percutaneous VX challenge are poorly understood. Percutaneously administered nerve agents are more slowly absorbed than inhaled agents; consequently, signs of poisoning occur later, with a longer duration. Telemetry was used to monitor heart rate, EEG, temperature and activity in guinea-pigs. Treatment with huBuChE at 30 or 120 min following percutaneous VX challenge ( approximately 2.5 x LD(50)) provided 100% protection from lethality. When huBuChE administration was delayed until the onset of observable signs of poisoning only 1 out of 6 animals survived to the end of the experiment at 7 days. This study adds to the body of evidence demonstrating the efficacy of huBuChE in animals by describing the successful therapeutic use of a protein bioscavenger as a post-exposure treatment against dermal exposure to VX up to 2h post-exposure. This study simultaneously used telemetric methods to show that the efficacy of huBuChE is linked to the prevention of detrimental physiological changes observed in control VX-treated animals. Post-exposure therapy is a promising additional indication for the concept of use of this material, and one that has particular relevance in a civilian exposure scenario.

Fc function of a new intravenous immunoglobulin product: IGIV 10% triple virally inactivated solution.

Background and Objectives  Baxter AG has developed a new liquid intravenous immunoglobulin product [Immune Globulin Intravenous (IGIV) 10%] using a new manufacturing procedure. A modified Cohn fractionation and ion exchange chromatography is used to produce an IgG solution with no alterations to the Fc region. Three dedicated virus reduction steps are included: solvent‐detergent treatment, nanofiltration, and incubation at low pH and elevated temperature in final formulation. We applied the reference method of the European Pharmacopoeia (EP) together with a flow‐cytometric binding assay for the evaluation of the Fc function of the new product.

Intravenous immunoglobulin (IVIg) Gammagard liquid contains anti-RAGE IgG and sLRP

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