Viktoria Weber

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.

Monocytes, peripheral blood mononuclear cells, and THP-1 cells exhibit different cytokine expression patterns following stimulation with lipopolysaccharide.

THP-1 cells are widely applied to mimic monocytes in cell culture models. In this study, we compared the cytokine release from THP-1, peripheral blood mononuclear cells (PBMC), monocytes, or whole blood after stimulation with lipopolysaccharide (LPS) and investigated the consequences of different cytokine profiles on human umbilical vein endothelial cell (HUVEC) activation. While Pseudomonas aeruginosa-stimulated (10 ng/mL) THP-1 secreted similar amounts of tumor necrosis factor alpha (TNF-α) as monocytes and PBMC, they produced lower amounts of interleukin(IL)-8 and no IL-6 and IL-10. Whole blood required a higher concentration of Pseudomonas aeruginosa (1000 ng/mL) to induce cytokine release than isolated monocytes or PBMC (10 ng/mL). HUVEC secreted more IL-6 and IL-8 after stimulation with conditioned medium derived from whole blood than from THP-1, despite equal concentrations of TNF-α in both media. Specific adsorption of TNF-α or selective cytokine adsorption from the conditioned media prior to HUVEC stimulation significantly reduced HUVEC activation. Our findings show that THP-1 differ from monocytes, PBMC, and whole blood with respect to cytokine release after stimulation with LPS. Additionally, we could demonstrate that adsorption of inflammatory mediators results in reduced endothelial activation, which supports the concept of extracorporeal mediator modulation as supportive therapy for sepsis.

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.

Removal of the Uremic Retention Solute p-Cresol Using Fractionated Plasma Separation and Adsorption

Removal of protein-bound uremic retention solutes, including p-cresol, by peritoneal dialysis and hemodialysis (HD) is limited. p-Cresol, mainly circulating as sulfate conjugate (p-cresyl sulfate [PCS]), is independently associated with mortality. Fractionated plasma separation and adsorption (FPSA) is a nonbiologic detoxification system for the treatment of liver failure. The FPSA clearance of uremic retention solutes is unknown. We studied PCS clearance by FPSA, using the Prometheus system. The neutral resin adsorbent and the anion exchange adsorbent bind PCS in vitro (reduction ratios [RRs] 37 and 70%). Ex vivo, the adsorbent mass removal (MR) (median 47.5 mg) contributes more than half to total MR (median 89.6 mg). In vivo, PCS RR during FPSA (50%) exceeded the RR during high flux HD (30%). We halted the study after four inclusions due to repeated thrombosis of the arterio-venous conduit. In conclusion, FPSA is a promising technique to improve clearance of protein-bound uremic retention solutes.

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept†

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.

Protective effect of resin adsorption on septic plasma-induced tubular injury

IntroductionA pro-apoptotic effect of circulating mediators on renal tubular epithelial cells has been involved in the pathogenesis of sepsis-associated acute kidney injury (AKI). Adsorption techniques have been showed to efficiently remove inflammatory cytokines from plasma. The aim of this study was to evaluate the efficiency of the hydrophobic resin Amberchrom CG161 M to adsorb from septic plasma soluble mediators involved in tubular injury.MethodsWe enrolled in the study 10 critically ill patients with sepsis-associated AKI and we evaluated the effects of their plasma on granulocyte adhesion, apoptosis and functional alterations of cultured human kidney tubular epithelial cells. We established an in vitro model of plasma adsorption and we studied the protective effect of unselective removal of soluble mediators by the Amberchrom CG161 M resin on septic plasma-induced tubular cell injury.ResultsPlasma from septic patients induced granulocyte adhesion, apoptosis and altered polarity in tubular cells. Plasma adsorption significantly decreased these effects and abated the concentrations of several soluble mediators. The inhibition of granulocyte adhesion to tubular cells was associated with the down-regulation of ICAM-1 and CD40. Resin adsorption inhibited tubular cell apoptosis induced by septic plasma by down-regulating the activation of caspase-3, 8, 9 and of Fas/death receptor-mediated signalling pathways. The alteration of cell polarity, morphogenesis, protein reabsorption and the down-regulation of the tight junction molecule ZO-1, of the sodium transporter NHE3, of the glucose transporter GLUT-2 and of the endocytic receptor megalin all induced by septic plasma were significantly reduced by resin adsorption.ConclusionsSeptic plasma induced a direct injury of tubular cells by favouring granulocyte adhesion, by inducing cell apoptosis and by altering cell polarity and function. All these biological effects are related to the presence of circulating inflammatory mediators that can be efficiently removed by resin adsorption with a consequent limitation of tubular cell injury.

Fluidized Bed Adsorbent Systems for Extracorporeal Liver Support

Abstract:  Acute liver failure based on acute‐on‐chronic liver failure (AoCLF) or on acute severe damage of the liver caused by different etiologies includes complex mechanisms resulting in severe disturbances of principle liver functions. In order to compensate the liver’s function of detoxification as efficiently as possible, fluidized bed absorbent systems have been designed. In these systems, small particles with specific adsorption properties for toxins related to acute liver failure are applied. A special technology based on adsorbents in suspension has been developed under the guidance of our group and is prepared for clinical application during the coming year. This technology is called microspheres‐based detoxification system (MDS) and is based on microadsorbents with a diameter of 1–10 µm which are recirculated in suspension. The safety of the MDS is guaranteed by the use of fluorescently labeled magnetic microparticles, which in case of a membrane‐leakage are detected in the blood circuit by an optical system equipped with a magnetic trap. In vitro tests with two kinds of microadsorbents (a combination of a hydrophobic neutral resin and an anion exchange resin) showed excellent efficiency of the system with respect to adsorption capacity as well as to the kinetics of elimination of albumin‐bound substances (e.g. unconjugated bilirubin or cholic acid) and of non‐protein‐bound substances (e.g. phenol). Moreover, using a plasma filter or the Albuflow filter as membrane filters in the blood circuit, the MDS technology offers the possibility to remove inflammatory mediators such as tumor necrosis factor‐α (TNF) by additional use of specific adsorbents.

Monitoring of endothelial cell activation in experimental sepsis with a two-step cell culture model

The aim of this work was to establish and characterize a cell culture model for lipopolysaccharide (LPS)-induced activation of human endothelial cells. Monocytic THP-1 cells were stimulated for 4 h with 10 ng/ml LPS from Pseudomonas aeruginosa in media containing 10% human plasma. Culture supernatants containing LPS and factors secreted by THP-1 in response to stimulation were applied to human umbilical vein endothelial cells (HUVECs). Nuclear factor-κB (NF-κB) activity, expression of adhesion molecules, and cytokine secretion were quantified. In addition, the effect of adsorptive removal of tumour necrosis factor-α (TNF-α) from the THP-1 culture supernatant on HUVEC activation was assessed. After 4 h of stimulation, THP-1 cells secreted various mediators including TNF-α (854 ± 472 pg/ml), interleukin (IL)-8 (2069 ± 710 pg/ml), IL-18 (305 ± 124 pg/ml), IL-10 (14 ± 5 pg/ml), and IL-1β (24 ± 11 pg/ml). Stimulated HUVECs showed significantly increased NF-κB activity and secreted high amounts of IL-6 and IL-8. Additionally, adhesion molecules ICAM-1 and E-selectin were increased both in the culture supernatant and at the cell surface. Removal of TNF-α from the THP-1 culture supernatant prior to HUVEC stimulation resulted in a decrease in NF-κB activity, expression of adhesion molecules, as well as IL-6 secretion. The cell culture model established in this study permits the monitoring of LPS-induced endothelial activation, which plays a central role in sepsis and may serve to assess the effect of mediator modulation by methods such as extracorporeal blood purification.

M2 Polarization of Human Macrophages Favors Survival of the Intracellular Pathogen Chlamydia pneumoniae.

Intracellular pathogens have developed various strategies to escape immunity to enable their survival in host cells, and many bacterial pathogens preferentially reside inside macrophages, using diverse mechanisms to penetrate their defenses and to exploit their high degree of metabolic diversity and plasticity. Here, we characterized the interactions of the intracellular pathogen Chlamydia pneumoniae with polarized human macrophages. Primary human monocytes were pre-differentiated with granulocyte macrophage colony-stimulating factor or macrophage colony-stimulating factor for 7 days to yield M1-like and M2-like macrophages, which were further treated with interferon-γ and lipopolysaccharide or with interleukin-4 for 48 h to obtain fully polarized M1 and M2 macrophages. M1 and M2 cells exhibited distinct morphology with round or spindle-shaped appearance for M1 and M2, respectively, distinct surface marker profiles, as well as different cytokine and chemokine secretion. Macrophage polarization did not influence uptake of C. pneumoniae, since comparable copy numbers of chlamydial DNA were detected in M1 and M2 at 6 h post infection, but an increase in chlamydial DNA over time indicating proliferation was only observed in M2. Accordingly, 72±5% of M2 vs. 48±7% of M1 stained positive for chlamydial lipopolysaccharide, with large perinuclear inclusions in M2 and less clearly bordered inclusions for M1. Viable C. pneumoniae was present in lysates from M2, but not from M1 macrophages. The ability of M1 to restrict chlamydial replication was not observed in M1-like macrophages, since chlamydial load showed an equal increase over time for M1-like and M2-like macrophages. Our findings support the importance of macrophage polarization for the control of intracellular infection, and show that M2 are the preferred survival niche for C. pneumoniae. M1 did not allow for chlamydial proliferation, but failed to completely eliminate chlamydial infection, giving further evidence for the ability of C. pneumoniae to evade cellular defense and to persist in human macrophages.

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.