Dieter Falkenhagen

Construction of a Functional S-Layer Fusion Protein Comprising an Immunoglobulin G-Binding Domain for Development of Specific Adsorbents for Extracorporeal Blood Purification

ABSTRACT The chimeric gene encoding a C-terminally-truncated form of the S-layer protein SbpA from Bacillus sphaericus CCM 2177 and two copies of the Fc-binding Z-domain was constructed, cloned, and heterologously expressed in Escherichia coli HMS174(DE3). The Z-domain is a synthetic analogue of the B-domain of protein A, capable of binding the Fc part of immunoglobulin G (IgG). The S-layer fusion protein rSbpA31-1068/ZZ retained the specific properties of the S-layer protein moiety to self-assemble in suspension and to recrystallize on supports precoated with secondary cell wall polymer (SCWP), which is the natural anchoring molecule for the S-layer protein in the bacterial cell wall. Due to the construction principle of the S-layer fusion protein, the ZZ-domains remained exposed on the outermost surface of the protein lattice. The binding capacity of the native or cross-linked monolayer for human IgG was determined by surface plasmon resonance measurements. For batch adsorption experiments, 3-μm-diameter, biocompatible cellulose-based, SCWP-coated microbeads were used for recrystallization of the S-layer fusion protein. In the case of the native monolayer, the binding capacity for human IgG was 5.1 ng/mm2, whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm2 was bound. This corresponded to 78 and 65% of the theoretical saturation capacity of a planar surface for IgGs aligned in the upright position, respectively. Compared to commercial particles used as immunoadsorbents to remove autoantibodies from sera of patients suffering from an autoimmune disease, the IgG binding capacity of the S-layer fusion protein-coated microbeads was at least 20 times higher. For that reason, this novel type of microbeads should find application in the microsphere-based detoxification system.

Neutral styrene divinylbenzene copolymers for adsorption of toxins in liver failure.

In artificial extracorporeal liver support systems, albumin-bound toxins such as bilirubin, bile acids, or aromatic amino acids are removed by adsorption to polymer beads. To overcome the potential weaknesses of anion exchange polymers currently used in liver support, namely, binding of heparin and activation of coagulation, we prepared two series of neutral polystyrene divinylbenzene resins with average pore sizes of 5-6 and 8-9 nm, respectively. In in vitro experiments using human plasma spiked with bilirubin, cholic acid, tryptophan, and phenol, we found that only pores larger than 5-6 nm were accessible to strongly albumin-bound substances, such as bilirubin. On the other hand, less strongly albumin-bound substances, such as bile acids, were efficiently bound by polymers of the small pore size range due to a higher accessible surface area. None of the neutral resins bound significant amounts of heparin. To assess the influence of the polymers on activation of coagulation, generation of thrombin-antithrombin complexes (TAT) was measured at different citrate concentrations. While none of the neutral polymers induced TAT generation, TAT levels were significantly elevated after incubation of plasma with an anion exchange polymer that is in clinical use for extracorporeal liver support. Binding characteristics of the neutral resins for the natural anticoagulants protein C and antithrombin showed remarkable differences, with weak binding of antithrombin but strong removal of protein C, not only for the anion exchanger, but also for neutral polymers of the large pore size range. In conclusion, neutral polystyrene divinylbenzene polymers with a pore size larger than 5-6 nm are efficient adsorbents for albumin-bound toxins that do not induce generation of thrombin-antithrombin complexes.

Development of Cationically Modified Cellulose Adsorbents for the Removal of Endotoxins

The removal of endotoxins by extracorporeal adsorption processes seems the most promising therapeutic approach to Gram-negative sepsis and endotoxin shock. However, thus far adsorbents have failed to bind endotoxins efficiently or have shown adverse biocompatibility characteristics. To overcome these disadvantages, small particles of regenerated cellulose in the range of 1-8 microns in diameter were produced. Before use, the microspheres were cationically modified by substitution with polyethyleneimine (PEI) or diethylaminoethyl (DEAE) groups. A third kind of adsorbent was manufactured by (physically) coating the cellulose matrix with PEI. All three types of adsorbents exhibited a high adsorption capacity for endotoxins in human plasma, whereas activated charcoal and various anion exchange resins removed only small amounts of endotoxins under the same conditions. In addition, because the outer surface area is very large, adsorption takes place rapidly and diffusion becomes almost irrelevant. The adsorption process is primarily based on electrostatic interactions, which could be demonstrated by a significantly higher adsorption rate and binding capacity for lipid A-diphosphoryl, compared with lipid A-monophosphoryl. Use of these adsorbents in a newly developed plasma sorption system could be of great clinical interest because of the low production costs, the high adsorption efficiency, and the excellent biocompatibility data.

Activation-dependent adsorption of cytokines and toxins related to liver failure to carbon beads.

In the course of severe pathological conditions, such as acute liver failure and sepsis, toxic metabolites and mediators of inflammation are released into the patients circulation. One option for the supportive treatment of these conditions is plasmapheresis, in which plasma, after being separated from the cellular components of the blood, is cleansed by adsorption of harmful molecules on polymers or activated carbon. In this work, the adsorption characteristics of activated carbon beads with levels of activation ranging from 0 to 86% were assessed for both hydrophobic compounds accumulating in liver failure (bilirubin, cholic acid, phenol and tryptophan) and cytokines (tumor necrosis factor α and interleukin-6). Progressive activation resulted in significant gradual reduction of both bulk density and mean particle size, in an increase in the specific surface area, and to changes in pore size distribution with progressive broadening of micropores. These structural changes went hand in hand with enhanced adsorption of small adsorbates, such as IL-6 and cholic acid and, to a lesser extent, also of large molecules, such as TNF-α.

Specific Blood Purification by Means of Antibody-Conjugated Magnetic Microspheres

The employment of immobilized antibodies (antigens) offers the most selective approach to extracorporeal adsorptive removal of humoral factors from blood. Polypeptide mediators and antibodies in systemic inflammatory and autoimmune diseases, respectively, can not be depleted by hemodialysis treatment, and thus represent attractive targets for immunoadsorption regimen. In this article we describe the elimination of interleukin 1β (IL-1β) and tumor necrosis factor α (TNFα), the major pathogenic mediators in septic shock and multi-organ failure, by superparamagnetic microspheres used in the novel MDS blood purification technology. The particles, consisting of antibody conjugates, exhibit high adsorption rate, capacity, selectivity, and biocompatibility. By application in an in vitro MDS set-up, human plasma could be efficiently cleansed from IL-ls and TNFa. Contrary to conventional blood and plasma chromatography techniques, MDS offers a high degree of flexibility and continuous blood processing due to magnetic adsorbent replacement.

Effect of anticoagulation with citrate versus heparin on the adsorption of coagulation factors to blood purification resins with different charge.

In liver failure, hydrophobic toxins accumulate in the blood circulation. To support hepatic function, extracorporeal blood purification systems have been developed, in which both cationic and neutral adsorbents are used to remove albumin-bound metabolites from blood. An issue of these systems is the additional removal of coagulation factors containing negatively charged γ-carboxyglutamate (Gla) domains, which, in physiological conditions, are shielded by calcium ions. We hypothesized that complexation of calcium ions by citrate leads to exposure of negative Gla domains, resulting in their binding to the positively charged adsorbents. The data presented here confirm that the binding of coagulation factors containing Gla domains to positively charged polymers is enhanced in the presence of citrate as compared to heparin. This effect increased with increasing charge density of the polymer and has important implications for the clinical application of positively charged polymers.