The removal of albumin bound substances has gained increasing interest in different diseases, especially in acute and chronic liver disease. Therefore, a new system, the fractionated plasma separation and adsorption (FPSA) system, was developed based on combined membrane and adsorbent blood purification techniques. The most important contribution to the FPSA system was the development of a new polysulfone hollow-fiber filter, which is characterized by a sieving coefficient of 0.89 for human serum albumin (HSA) but only of 0.17 for fibrinogen, and 0 (zero) for IgM immunoglobulins. Using a closed filtrate circuit connected to the new polysulfone filter which integrates 1 or 2 adsorption columns and also a high flux dialyzer adapted to a dialysis machine, the FPSA system opens excellent possibilities for the relatively specific removal of albumin bound substances from the blood such as albumin bound bilirubin or even tryptophan. In comparison to other systems (for example, the Molecular Adsorbent Recirculating System [MARS] and albumin dialysis systems), the FPSA system enables much higher elimination of strongly bound albumin substances. The first clinical investigations have recently started based on a modified dialysis machine designed with all necessary safety measures.
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 rSbpA 31-1068 /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-m-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/mm 2 , whereas after cross-linking with dimethyl pimelimidate, 4.4 ng of IgG/mm 2 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.
Background: The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence-activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis.Methods: Mononuclear cells were collected from 10 L of blood by leukapheresis, to which carboxyfluorescein succinimidyl ester prelabeled CaOV-3 tumor cells were spiked at a ratio of 26 to 10 6 leukocytes. Elutriation separated the spiked leukapheresates primarily by cell size into distinct fractions, and leukocytes and tumor cells, characterized as carboxyfluorescein succinimidyl ester positive, EpCAM positive and CD45 negative events, were quantified by flow cytometry. Tumor cells were isolated from the last fraction using FACS or anti-EpCAM coupled immunomagnetic beads, and their recovery and purity determined by fluorescent microscopy and real-time PCR.Results: Leukapheresis collected 13.5 3 10 9 mononuclear cells with 87% efficiency. In total, 53 to 78% of spiked tumor cells were pre-enriched in the last elutriation fraction among 1.6 3 10 9 monocytes. Flow cytometry predicted a circulating tumor cell purity of $90% giving an enrichment of 100,000-fold following leukapheresis, elutriation, and FACS, where CaOV-3 cells were identified as EpCAM positive and CD45 negative events. FACS confirmed this purity. Alternatively, immunomagnetic bead adsorption recovered 10% of tumor cells with a median purity of 3.5%.Conclusions: This proof of concept study demonstrated that elutriation and FACS following leukapheresis are able to enrich and isolate tumor cells from a large blood volume for molecular characterization.
The extent to which bacterial products from contaminated dialysate enter a patient's blood depends upon the type and permeability of the hemodialysis membrane in use. This study was performed to assess the transfer of pyrogenic substances across both high- and low-flux membranes (DIAPES, Fresenius Polysulfone, Helixone, Polyamide S). All experiments were carried out in the saline-saline model. The dialysate pool was contaminated either with purified lipopolysaccharide (LPS) (250 and 500 EU/mL) or with sterile bacterial culture filtrates (20 EU/mL), and in vitro dialysis was performed under diffusive and convective conditions. A significant transfer of endotoxin was observed for both low- and high-flux DIAPES challenged with either LPS or with bacterial culture filtrates. Under identical conditions, no transfer of endotoxins was detectable across Fresenius Polysulfone and Helixone upon challenge with purified LPS. With bacterial culture filtrates, endotoxin concentrations for Polyamide S and Fresenius Polysulfone were about 10% and 1%, respectively, of those measured for DIAPES, whereas no transfer of endotoxin was detectable for Helixone. Using an alternative assay (induction of interleukin-1 receptor antagonist, IL-1Ra, in whole blood), only the DIAPES membrane showed the passage of cytokine-inducing substances. Thus, when saline is present in both the blood and dialysate compartments (i.e., the situation during predialysis priming procedures), dialysis membranes differ profoundly with respect to their permeability to endotoxins.
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.
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