Bai Hu Tang, Si Ni Tang, and Xue Bi Tang amplify pro-inflammatory activities and reduce apoptosis in endothelial cells in a cell culture model of sepsis.

ETHNOPHARMACOLOGICAL RELEVANCE: Sepsis is a systemic inflammatory response of the body to a severe infection or massive tissue injury. Despite intensive research, sepsis continues to have a high mortality rate and successful treatment options are strongly needed. Bai Hu Tang (BHT), Si Ni Tang (SNT), and Xue Bi Tang (XBT) are ancient traditional Chinese formulas derived from Chinese herbs that are used to treat Sepsis, but their mechanisms of activity are largely unknown. AIM OF THE STUDY: We aimed to examine dose-dependent effects of BHT, SNT, and XBT in a cell culture model of Sepsis, with special focus on endothelial cell apoptosis and the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)6, IL8, the surface adhesion molecule intercellular adhesion molecule-1 (ICAM-1) and endothelial-leukocyte adhesion molecule-1 (ELAM-1). MATERIAL AND METHODS: We stimulated THP1 monocytic cells with lipopolysaccharide (LPS, Escherichia coli (E. coli)) for 4 h and used the resulting culture medium to stimulate human umbilical vein endothelial cells (HUVECs). HUVECs were also simultaneously treated with hydrophilic concentrates of BHT, SNT or XBT. We evaluated the mRNA and protein expression levels of IL6, IL8, MCP-1, ICAM-1, and ELAM-1 and the activity of caspase 3/7, a marker of cell apoptosis, after stimulation and treatment. In addition, we stimulated cannulated veins from human umbilical cords for 24 h and treated them with BHT, SNT or XBT. Immunohistochemistry visualized expression of ICAM-1 and ELAM-1. RESULTS: The mRNA and protein levels of IL6, IL8, ICAM-1, and ELAM-1 were higher in stimulated HUVECs than in controls. Treating stimulated HUVECs with BHT, SNT or XBT induced an additional increase in IL6 (13- to 132-fold) and IL8 (17- to 32-fold) mRNA levels but did not influence their protein levels. In addition, BHT induced an additional increase in ICAM-1 mRNA (9-fold) expression, whereas XBT increased the mRNA and protein levels of ELAM-1 by 42-fold and 10-fold, respectively. Finally, caspase 3/7 levels, and therefore apoptosis, were up to 100% lower in cells treated with BHT than in the stimulated control (P < 0.001). CONCLUSION: The results of this study indicate that BHT, SNT, and XBT interfere in inflammatory pathways during septic processes by reducing the apoptotic effects of LPS and modifying the endothelial expression of pro-inflammatory cytokines and surface adhesion molecules.

miR-146a, miR-146b, and miR-155 increase expression of IL-6 and IL-8 and support HSP10 in an In vitro sepsis model.

microRNAs (miRNAs) play an essential role in inflammation processes including sepsis. This study aimed to identify miRNAs as candidates for therapies that are involved in the innate immune response and to assess their potential functions in the activation of the endothelium. We stimulated THP-1 monocytes with 10 ng/ml LPS for 4 h and used the supernatant for the stimulation of human umbilical vein endothelial cells (HUVEC) or human pulmonary microvascular endothelial cells (HPMEC) for 16 h. miRNA array analysis (of 1,891 miRNAs) identified a 1.5-fold upregulation of miR-146a, miR-146b, and miR-155 in stimulated endothelial cells. HUVEC were transfected with miRNA inhibitors for miR-146a, miR-146b, and miR-155 to investigate the function of these miRNAs in endothelial inflammatory pathways. Inhibition of miR-146a resulted in a diminished release of interleukin (IL)-6 and IL-8 by respective 68% and 55% (P<0.001). Inhibition of miR-146b reduced the expression of IL-6 by 49% (P<0.001). Inhibition of miR-155 reduced the expression of IL-6 and IL-8 by respective 31% (P<0.001) and 14%. The inhibition of miR-146a, miR-146b, and miR-155 reduced the release of HSP10 by 50%, 35%, and 69% (P<0.05), respectively, but did not influence the expression of HSP27 or TXA2. In conclusion, miR-146a, miR-146b, and miR-155 are exerting anti-inflammatory properties by down-regulating IL-6 and IL-8, and influencing the expression of HSP10 in the activated endothelium. We provide evidence for the central role of selected miRNAs in sepsis and their use in the development of small interfering RNA therapeutics to target immune cells and sepsis pathways.

Pore size--a key property for selective toxin removal in blood purification.

PURPOSE: Extracorporeal blood purification systems based on combined membrane/adsorption technologies are used in acute liver failure to replace detoxification as well as to remove inflammatory mediators in sepsis patients. In addition to coating and chemical modification of the surface, pore size significantly controls the selectivity of adsorption materials. METHODS: This study addresses the adsorption of albumin bound liver toxins, cytokines, and representative plasma compounds on three adsorbents which differ only in pore size distribution. All three adsorbents are based on hydrophobic poly(styrene-divinylbenzene) copolymer matrices and have mean pore sizes of 15, 30, and 100 nm. RESULTS: The pores of adsorbents act as molecular sieves and prevent the entry of molecules that are larger than their molecular cut-off. The results of this study reveal that adsorbents based on styrene-divinylbenzene polymers with 15 nm pores are suitable for cytokine removal, and the same adsorbents with 30-40 nm pores are the best choice for the removal of albumin-bound toxins in the case of liver failure. Adsorbents with very large pores lack selectivity which leads to uncontrolled adsorption of all plasma proteins. Therefore, hydrophobic adsorbents with large pores offer inadequate plasma compatibility and do not fulfill the requirements for blood purification. CONCLUSIONS: Biocompatibility and efficiency of adsorbents used for blood purification can improved by fine tuning of adsorbent surface pore distributions.

Endotoxin adsorbents in extracorporeal blood purification: do they fulfill expectations?

INTRODUCTION: Lipopolysaccharides (LPS) are extremely strong stimulators of inflammatory reactions, act at very low concentrations, and are involved in the pathogenesis of sepsis and septic shock. Because of its toxicity, the efficient removal of endotoxin from patients' blood is very important in clinical medicine. The purpose of this study was to determine the endotoxin adsorption capacities of commercial endotoxin adsorbers for endotoxin removal in buffer solution, protein solution, serum and heparinized plasma; some of these were also characterized in whole blood. The tested LPS adsorbers were Toraymyxin® PMX-20R, Alteco® LPS Adsorber, DEAE-Sepharose, Polymyxin B-Agarose, and EndoTrap® red. METHODS: The adsorber materials were tested in buffer and protein solutions spiked with fluorescently labeled LPS (100 ng/ml). Additionally, batch tests with LPS-spiked serum, heparinized plasma and whole blood were performed with an LPS concentration of 5 ng/ml. Additionally, the washing solutions of the two tested Polymyxin B (PMB)-based adsorbers were analyzed for PMB release to determine if the resulting LPS inactivation was caused by PMB leakage. RESULTS: The results show that DEAE-Sepharose was most effective in LPS adsorption. Of the other tested endotoxin removal materials, only the PMB-based adsorbers were able to reduce the LPS activity. However, we were able to show that the reduction in LPS activity was caused by desorbed PMB, which inactivates endotoxins. CONCLUSIONS: None of the adsorbents that were tested in this study showed promising results for potential use in extracorporeal blood purification.

A target-oriented algorithm for citrate-calcium anticoagulation in clinical practice.

BACKGROUND/AIMS: Because of a high monitoring demand and an ensuing need for automation of regional citrate anticoagulation (RCA), a new semi-automated target-oriented algorithm was developed. The aim of this study was the evaluation of its functionality and safety. METHODS: Fourteen haemodialysis patients were treated 5 times consecutively with RCA. Samples were drawn pre- and post-infusion once per hour. Electrolytes, blood cell counts, acid-base and coagulation parameters were analyzed. RESULTS: Mean ionized calcium (Ca(2+)) values pre-filter were 0.23 and 0.33 mmol/l in the 0.2 and 0.3 mmol/l target groups, respectively. Extraction ratios for citrate and total calcium through the dialysis filter were constant during the entire treatment (83 and 68%, respectively). Citrate accumulation was avoided. CONCLUSION: The new algorithm enables safe and accurate RCA. By regulating Ca(2+) pre-filter using the target-oriented algorithm, the degree of anticoagulation may be easily controlled.

20 years since the establishment of the BANTAO association (Balkan Cities Association of Nephrology, Dialysis, Transplantation and Artificial Organs).

The Balkan Cities Association of Nephrology, Dialysis, Transplantation and Artificial Organs (BANTAO) was born in Ohrid on October 9, 1993. The war in former Yugoslavia negatively affected the development of nephrology and also the connections among the nephrologists from the Balkans. However, there was willingness for further mutual collaboration between the nephrologists from the Balkans. The war in Yugoslavia created hate among people, between the newly established countries, and there were problems with the recognition of the names of the new countries, and so, the nephrologists decided to apply the ancient principle of using the names of the cities, instead of the countries, as the founders of the Association. The main goal of BANTAO is to promote scientific and technical cooperation in the fields of renal disease and artificial organs between the regions on the Balkan Peninsula and the world, to give an opportunity for exchange of experience and knowledge among the experts in the area and to engage in collaborative projects in order to demonstrate that cooperation is possible even on the turbulent Balkan Peninsula. The I BANTAO congress was held in Varna from September 22 to 24th, 1995 (President--D. Nenov, Varna). The II congress of BANTAO was held from September 6th to 10th, 1997 in Struga, (President--M. Polenakovic, Skopje). The III BANTAO congress was held in Belgrade from September 18th to 20th, 1998 (President--Lj. Djukanovic, Belgrade). The IV congress of BANTAO was held in Izmir from 14th to 16th November 1999 (President--A. Akcicek, Izmir). The V Congress of BANTAO was held in Thessaloniki from September 30th to October 3rd, 2001 (President--P. Stathakis, Athens). The VI Congress of BANTAO was held for the second time in Varna from 6th to 9th October 2003 (President--D. Nenov, Varna). The VII congress of BANTAO was held from September 8th to 11th, 2005 in Ohrid, (President--M. Polenakovic, Skopje). The VIII BANTAO congress was held in Belgrade, 16-19 September 2007 (President--V. Nesic, Belgrade). The IX BANTAO congress was held in Antalya, 18-22 November 2009 (President--A. Basci, Izmir). The X BANTAO congress was held from 13 to 15 October 2011 in Chalkidiki (President--D. Tsakiris, Thessaloniki). The XI BANTAO congress is being held on 26-29 September 2013 in Timisoara (President--A. Schiller, Timisoara). At the VII BANTAO Congress for the first time a CME Course was organized by ERA/EDTA and ISN/COMGAN entitled "Frontiers in Nephrology" with seven distinguished speakers. Very important event in the existence of BANTAO is the appearance of the BANTAO journal in 2003. The BANTAO journal has been published biannually since 2003. In the past 10 years, 20 regular issues; 2 supplements (Antalia and Chalkidiki congresses) have been published. Editors of the journal were as follows: 2003-2005--D. Nenov, Editor; 2005-2009--A. Basci, Editor; 2009--Goce Spasovski, Editor. Until now 332 papers have been published. The BANTAO journal is on EBSCO, DOAJ, SCOPUS. After the First Congress of BANTAO, F. Valderrábano, chairman of the EDTA--ERA Registry, at that time, wrote in Nephrology Dialysis Transplantation (1996) 11:740: "Nephrologists of the Balkan countries meet across political frontiers and war fronts--an example to politicians! BANTAO: a new European Medical Association overcomes Political obstacles." Despite the difficulties imposed by major events, such as devastating wars and catastrophic earthquakes in many countries of the Balkan Peninsula BANTAO has made considerable progress. The BANTAO Congress was established as the major scientific and institutional forum for Balkan nephrologists, with its own journal, indicating our will to communicate, to collaborate, to get to know each other and to share our difficulties. Now, we expect further successful work of BANTAO.

In vitro investigations of citrate clearance with different dialysis filters.

OBJECTIVE: In extracorporeal blood purification, citrate anticoagulation offers several substantial advantages over conventional heparin anticoagulation. However, there is still a lack of information on citrate kinetics, especially on the citrate clearance of conventional hemodialyzers. The aim of this study was to investigate the citrate clearance for different hemodialysis filters as a basis for the development of an intelligent citrate-calcium infusion algorithm. MATERIALS AND METHODS: For our experiments, the Fresenius 4008H dialysis machine and the dialysis filters FX 60, F6 HPS, F8 HPS (Fresenius Medical Care, Bad Homburg, Germany), Polyflux 140H and 14L (Gambro Holding, Stockholm, Sweden), Xenium 130 (Baxter AG, Vienna, Austria) and APS-650 (ASAHI Kasei Kuraray Medical, Chiyoda-ku, Japan) were used. Clearance calculations were performed based on plasma/blood flow rate and the citrate concentrations at filter inlet and outlet. All experiments were carried out in vitro with fresh frozen plasma (FFP) or whole blood. RESULTS: The results prove that citrate clearance is significantly higher with high-flux filters than with low-flux filters. Higher dialysate flow rates cause a more effective removal of citrate. The citrate clearance for low-flux and high-flux filters was 71 ± 7 and 86 ± 1% of the urea clearance, respectively. CONCLUSIONS: Citrate can efficiently be removed with standard hemodialysis. However, depending on the infused amounts as well as on the patient - especially in patients with impaired liver function - the use of a high-flux dialysis filter and a high dialysate flow rate should be considered to minimize the risk of citrate accumulation.

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.

Adsorptive modulation of inflammatory mediators dampens endothelial cell activation.

AIM: In this study, the effect of specific or selective adsorption of inflammatory mediators on endothelial activation was assessed. METHODS: Conditioned medium was obtained by stimulation of monocytic THP-1 cells with lipopolysaccharide and treated either with an adsorbent specific for tumour necrosis factor-α or with an albumin-coated polystyrene-divinylbenzene copolymer which selectively binds a range of cytokines. Thereafter, the conditioned medium was applied to endothelial cells in culture. RESULTS: Adsorption of inflammatory mediators resulted in significantly decreased endothelial cell activation, as shown by reduced interleukin (IL)-6 and IL-8 secretion from endothelial cells as well as reduced surface expression of the adhesion molecules intercellular adhesion molecule-1 and E-selectin. The effect was more pronounced the earlier the mediator modulation was performed. CONCLUSION: Adsorptive modulation of inflammatory mediators dampens endothelial cell activation and may thus be beneficial as supportive therapy in sepsis.

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 patient's 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.

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

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 106 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 x 109 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 x 109 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.

Characterization of SVEP1, KIAA, and SRPX2 in an in vitro cell culture model of endotoxemia.

To assess the influence of unknown factors in endotoxemia, a conditioned medium, achieved by the stimulation of THP1 monocytes with lipopolysaccharide (LPS) [4h], was used for the stimulation of human umbilical vein endothelial cells (HUVECs) [16h]. SVEP1, KIAA0247, and SRPX2 were selected after microarray analysis. To study their possible functions, siRNAs of SVEP1, KIAA0247, or SRPX2 were used for the transfection of HUVECs and cells were stimulated with conditioned medium [16h]. Inhibition of SVEP1 expression resulted in an increase of soluble intercellular adhesion molecule (sICAM) 1 (10%) and soluble E-selectin (sE-selectin) (19%). Inhibition of SRPX2 led to an increase of sICAM (11%) and sE-selectin (14%). KIAA0247 negative HUVECs showed a decrease in monocyte chemoattractant protein (MCP) 1 of 16%. SVEP1 and SRPX2 seemed to act as regulators of ICAM1 and E-selectin shedding and influence the expression of membrane bound adhesion molecules.

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-1ß (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.

Alginate-encapsulated human hepatoma C3A cells for use in a bioartificial liver device - the hybrid-MDS.

PURPOSE: The aim of this study was to encapsulate C3A cells into alginate microcapsules with an average diameter of < or =100 microm, thus enabling them to be recirculated in a bioartificial liver device based on MDS (Microsphere-based Detoxification System) technology. The microcapsules have to be permeable for essential proteins such as albumin. METHODS: C3A cells were encapsulated using alginate. The resulting alginate beads were coated with poly(diallyldimethylammoniumchloride) (pDADMAC) and poly(sodium-p-styrenesulfonate) (pSS). Their mechanical stability was tested by recirculation of the microcapsule suspension, while their permeability was determined by reverse-size exclusion chromatography and by the use of a confocal laser microscope. The metabolic activities of encapsulated C3A cells were compared to freely growing adherent C3A cells in static cultivation models. The metabolic functionality of encapsulated C3A cells in static conditions was compared to encapsulated C3A cells in a dynamic model. RESULTS: The mean diameter of the resulting microcapsules was 86 mum. Our experiments show that these microcapsules were permeable for albumin and the high flow rate of 600 ml/min in a dynamic model has no influence on the survival and the metabolic activities of the encapsulated cells during the tested time of 24 hours. CONCLUSIONS: Alginate microcapsules containing C3A cells can be used to produce albumin and growth factors in a bioartificial or hybrid liver support system. Thanks to their small diameter, the microcapsules in suspension can be recirculated in the MDS.

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.

Magnetic fluorescent microparticles as markers for particle transfer in extracorporeal blood purification.

The microspheres-based detoxification system (MDS) is a combined membrane-adsorption system for extracorporeal blood purification in which adsorbent microparticles are recirculated in an extracorporeal filtrate circuit. Because the plasma filter represents the only barrier between the adsorbents and the patient's blood, there is the potential risk of particle entrance into the patient in case of a membrane rupture. To guarantee first fault safety of the system required for clinical application, magnetic fluorescent microparticles are added as markers to the adsorbent circuit. Detection of these particles in the venous blood line results in immediate shutdown of the pumps. Magnetic beads were functionalized with cresyl violet and tested with an in vitro setup of the particle detector to assess the detection limit in different matrices (water versus blood) as well as the influence of flow rate and particle size on the signal. In addition, biocompatibility and influence of sterilization on the performance of the particles were assessed. Functionalization of the magnetic particles with cresyl violet yielded fluorescent particles that were stable at 4 degrees C for at least 12 months. No leakage of dye was detectable, and the particles were neither cytotoxic nor mutagenic. The particles could be steam sterilized without significant loss in fluorescence intensity. With an in vitro setup of the particle detector, 0.1 mg and 5 mg of particles were reproducibly detectable in water and blood, respectively.

Citrate anticoagulation control by ionized calcium levels does not prevent hemostasis and complement activation during hemodialysis.

The purpose of this study was to determine whether or not regional citrate anticoagulation (RCA) controlled by ionized calcium (iCa(2+)) would overcome thrombogenicity, prevent hemostasis, and complement activation during hemodialysis (HD). RCA was performed in 10 patients during 10 HD sessions using a polysulfone membrane in an effort to keep iCa(2+) at dialyzer outlet at < or =0.4 mmol/L. Compared to baseline, plasma levels of thrombin-antithrombin III complexes rose significantly at 240 min, and tissue factor and complement C5a component levels at 30 and 240 min of the procedure. Thrombocyte count declined significantly at 30 and 240 min, while activated clotting time (ACT) did not increase significantly, and platelet factor 4 as well as von Willebrand factor levels did not alter significantly. While ACT correlated significantly with some thrombogenicity markers, iCa(2+) did not correlate with ACT, changes in hemostasis, or C5a. We conclude the usually recommended iCa(2+) levels in the HD extracorporeal circuit did not guarantee the complete overcoming of thrombogenicity, prevention of hemostasis, and complement activation.

Efficient adsorption of tumor necrosis factor with an in vitro set-up of the microspheres-based detoxification system.

BACKGROUND: The Microsperes-Based Detoxification System (MDS) represents a flexible therapeutic option for various diseases, depending on the specificity of the adsorbents applied. A potential application of the MDS is the supportive therapy of sepsis. METHODS: Microadsorbents for tumor necrosis factor (TNF) were prepared by immobilization of anti-TNF antibodies on cellulose (1-10 microm) and applied in an experimental set-up using a pool of human plasma (1 liter) spiked with TNF (800 pg/ml) and its soluble receptors (1,000 pg/ml each). Removal of TNF was compared using a plasma filter and the albumin-permeable filter Albuflow. RESULTS: Addition of 4 (2) g of adsorbent to the filtrate circuit reduced TNF concentrations in the pool by 80% (64%). Removal rates did not differ significantly for the different filters. TNF adsorption was not influenced by the presence of excess TNF receptors. CONCLUSIONS: Concentrations of mediators can be efficiently modulated with the MDS using small quantities of specific adsorbents.

Fluidized bed adsorbent systems for extracorporeal liver support.

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 microm 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-alpha (TNF) by additional use of specific adsorbents.