Looking beyond endotoxin: a comparative study of pyrogen retention by ultrafilters used for the preparation of sterile dialyis fluid

Sterile single-use ultrafilters are used in dialysis for the preparation of the substitution fluid given to patients undergoing dialysis treatments with high convective fluid removal. The retention of pyrogenic agents by the ultrafilters is crucial to avoiding inflammatory responses. The performance of a new single-use ultrafilter (NUF) with a positively charged flat sheet membrane of relatively small membrane area and large pore size was compared to a reference ultrafilter (RUF) with a hollow fiber membrane. Filter performance was tested with various pyrogen-contaminated dialysis fluids by direct pyrogen quantification and by measuring inflammatory responses in cell-based bioassays. The NUF completely retained oligodeoxynucleotides (ODN), whereas the RUF was fully permeable. Both filters tended to decrease biological activity of DNA in filtered bacterial lysates. The NUF reduced lipopolysaccharides (LPS) and LPS-induced biological activity by 100%, whereas the RUF produced filtrates with low but detectable levels of LPS in most cases. Peptidoglycans (PGN) were fully retained both by the NUF and the RUF. The new ultrafilter retained biologically active ODN, which has not yet been described for any other device used in dialysis, and it showed better or equal retention of LPS and PGN even with a smaller membrane surface and larger pore size

Medium Cut-Off (MCO) Membranes Reduce Inflammation in Chronic Dialysis Patients—A Randomized Controlled Clinical Trial

Background To increase the removal of middle-sized uremic toxins a new membrane with enhanced permeability and selectivity, called Medium Cut-Off membrane (MCO-Ci) has been developed that at the same time ensures the retention of albumin. Because many middle-sized substances may contribute to micro-inflammation we hypothesized that the use of MCO-Ci influences the inflammatory state in hemodialysis patients. Methods The randomized crossover trial in 48 patients compared MCO-Ci dialysis to High-flux dialysis of 4 weeks duration each plus 8 weeks extension phase. Primary endpoint was the gene expression of TNF-α and IL-6 in peripheral blood mononuclear cells (PBMCs), secondary endpoints were plasma levels of specified inflammatory mediators and cytokines. Results After four weeks of MCO-Ci the expression of TNF-α mRNA (Relative quantification (RQ) from 0.92 ± 0.34 to 0.75 ± 0.31, -18.5%, p<0.001)-α and IL-6 mRNA (RQ from 0.78 ± 0.80 to 0.60 ± 0.43, -23.1%, p<0.01) was reduced to a significantly greater extent than with High-flux dialyzers (TNF mRNA-RQ: -14.3%; IL-6 mRNA-RQ: -3.5%). After retransformation of logarithmically transformed data, measurements after MCO were reduced to 82% of those after HF (95% CI 74%–91%). 4 weeks use of MCO-Ci resulted in long- lasting change in plasma levels of several cytokines and other substances with a significant decrease for sTNFR1, kappa and lambda free light chains, urea and an increase for Lp-PLA2 (PLA2G7) compared to High-flux. Albumin levels dropped significantly after 4 weeks of MCO dialysis but increased after additional 8 weeks of MCO dialysis. Twelve weeks treatment with MCO-Ci was well tolerated regarding the number of (S)AEs. In the extension period levels of CRP, TNF-α-mRNA and IL-6 mRNA remained stable in High-flux as well as in MCO-Ci. Conclusions MCO-Ci dialyzers modulate inflammation in chronic HD patients to a greater extent compared to High-flux dialyzers. Transcription of pro-inflammatory cytokines in peripheral leukocytes is markedly reduced and removal of soluble mediators is enhanced with MCO dialysis. Serum albumin concentrations stabilize after an initial drop. These results encourage further trials with longer treatment periods and clinical endpoints

Assessment of the association between increasing membrane pore size and endotoxin permeability using a novel experimental dialysis simulation set-up

Background: Membranes with increasing pore size are introduced to enhance removal of large uremic toxins with regular hemodialysis. These membranes might theoretically have higher permeability for bacterial degradation products. In this paper, permeability for bacterial degradation products of membranes of comparable composition with different pore size was investigated with a new in vitro set-up that represents clinical flow and pressure conditions. Methods: Dialysis was simulated with an AK200 machine using a low-flux, high-flux, medium cut-off (MCO) or high cut-off (HCO) device (n = 6/type). A polyvinylpyrrolidone-solution (PVP) was recirculated at blood side. At dialysate side, a challenge solution containing a filtrated lysate of two water-borne bacteria (Pseudomonas aeruginosa and Pelomononas saccharophila) was infused in the dialysate flow (endotoxin >= 4EU/ml). Blood and dialysate flow were set at 400 and 500 ml/min for 60 min. PVP was sampled before (PVPpre) and after (PVPpost) the experiment and dialysate after 5 and 55 min. Limulus Amebocyte Lysate (LAL) test was performed. Additionally, samples were incubated with a THP-1 cell line (24 h) and IL-1 beta levels were measured evaluating biological activity. Results: The LAL-assay confirmed presence of 9.5 +/- 7.4 EU/ml at dialysate side. For none of the devices the LAL activity in PVPpre vs. PVPpost was significantly different. Although more blood side PVP solutions had a detectable amount of endotoxin using a highly sensitive LAL assay in the more open vs traditional membranes, the permeability for endotoxins of the 4 tested dialysis membranes was not significantly different but the number of repeats is small. None of the PVP solutions induced IL-1 beta in the THP-1 assay. Conclusions: A realisitic in vitro dialysis was developed to assess membrane translocation of bacterial products. LAL activity on the blood side after endotoxin exposure did not change for all membranes. Also, none of the PVPpost solutions induced IL-1 beta in the THP-1 bio-assay

The Dialyzer as the Last Line of Protection against Endotoxins

When dialysis fluid is contaminated with endotoxins, the dialyzer membrane is often referred to as the last line of protection to prevent endotoxins from entering the patient’s blood. However, a quantifiable requirement for this endotoxin retention property of the membrane has not yet been defined. The ANSI/AAMI/ISO 23500 standard series provides the framework for the microbiological quality of dialysis water, concentrates, and dialysis fluid, and defines the limit value for the non-pyrogenic endotoxin dose. After defining the boundary conditions of the endotoxin loading of the membrane by dialysis fluid and the patient’s non-pyrogenic endotoxin dose, quantifiable requirements for the endotoxin retention properties of a membrane, expressed as a dimensionless logarithmic retention value (LRV), were developed in this work. Based on standard dialysis fluid quality, the LRV should minimally be two for a protein-coated membrane after contact with patient blood and minimally be one for a protein-free pristine membrane during online priming before contact with patient blood. This work also presents the critical factors for endotoxin retention tests and shows that the defined LRV values are reached by membranes in modern dialyzers

Cytochrome C Biosensor—A Model for Gas Sensing

This work is about gas biosensing with a cytochrome c biosensor. Emphasis is put on the analysis of the sensing process and a mathematical model to make predictions about the biosensor response. Reliable predictions about biosensor responses can provide valuable information and facilitate biosensor development, particularly at an early development stage. The sensing process comprises several individual steps, such as phase partition equilibrium, intermediate reactions, mass-transport, and reaction kinetics, which take place in and between the gas and liquid phases. A quantitative description of each step was worked out and finally combined into a mathematical model. The applicability of the model was demonstrated for a particular example of methanethiol gas detection by a cytochrome c biosensor. The model allowed us to predict the optical readout response of the biosensor from tabulated data and data obtained in simple liquid phase experiments. The prediction was experimentally verified with a planar three-electrode electro-optical cytochrome c biosensor in contact with methanethiol gas in a gas tight spectroelectrochemical measurement cell

Evolution of double psi beta-barrel proteins und aspartic proteases

In drei Schwerpunkten wurde die Evolution der Double psi beta-barrel Proteine und Aspartatproteasen untersucht. Im ersten Schwerpunkt ging es um die Faltungen Double psi beta-barrel, deren Vertreter als N-terminale Domänen von AAA-ATPasen wie Vat oder Cdc48 vorkommen, und Acid proteases, zu der die Aspartatproteasen Pepsin und HIV-1 Protease gehören. Obwohl die Strukturen dieser Faltungen bereits als sehr ähnlich erkannt und beschrieben wurden, werden sie bisher als analoge Strukturen mit konvergenter Evolution beschrieben. Dies stützte sich vor allem auf die fehlende Sequenzähnlichkeit und geringe Unterschiede der topologischen Verknüpfung ihrer beta-Stränge. In dieser Arbeit sollten funktionelle Untersuchungen Hinweise auf eine mögliche gemeinsame Abstammung dieser Faltungen liefern. Dazu wurden Chaperonassays durchgeführt, welche Aggregationsverhinderung als funktionelle Gemeinsamkeit von Vertretern dieser Faltungen offenbarten. Die Ergebnisse bestärkten die Vermutung, die Vertreter dieser Faltungen als entfernte Verwandte zu betrachten. Damit wurde ein weiteres Beispiel geliefert, wie sich eine Faltung durch divergente Evolution ändern kann und dies zu verwandten Proteinfaltungen führt. Ein zusätzlicher Aspekt in diesem Schwerpunkt war die Frage, ob es möglich sei, Domänen dieser Faltungen in ihrem Domänenkontext gegeneinander auszutauschen. Im zweiten Schwerpunkt wurde die Verwandtschaft der beiden Proteinfaltungen Double psi beta-barrel und Swapped hairpin-barrel untersucht. Die Verwandtschaft wurde aufgrund von markanten Strukturähnlichkeiten bereits vermutet. Die Sequenzähnlichkeit ist jedoch sehr gering und hinterließ Unsicherheit. Die Verwandtschaft dieser Faltungen wäre ein weiteres Beispiel für einen Faltungswechsel durch divergente Evolution. Der Hypothese über die homologen Faltungen fehlte jedoch ein plausibler Evolutionsmechanismus, der die Entstehung durch bekannte genetische Mechanismen beschreiben würde. In dieser Arbeit wurden die Strukturen der Proteine PhS018 und Af1504 untersucht. Durch Aufklärung der Struktur des Proteins PhS018 von Pyrococcus horikoshii konnte ein weitere, neue Faltung beschrieben werden: die Faltung RIFT-barrel. Sie steht intermediär zwischen Double psi beta-barrel und Swapped hairpin-barrel und kann die Entstehung dieser beiden Faltungen erklären. Darüber hinaus wurde durch Identifikation eines gemeinsamen, strukturell hoch konserviertes beta/alpha/beta-Elements die Evolution dieser Faltungen aus einfachen Supersekundärstrukturmotiven beschrieben werden. Neben PhS018 wurde das potentielle Double psi beta-barrel Protein Af1504 von Archaeoglobus fulgidus biochemisch untersucht. Im dritten Schwerpunkt wurde die Funktion eines Operons aus Archaeoglobus fulgidus untersucht. In diesem Operon wird das Protein Af1504 zusammen mit den Proteinen Af1502, Af1503 und Af1505 kodiert. Zum Operon gehört das Transmembranprotein Af1503, für das bereits eine cytoplasmatische HAMP Domäne vorhergesagt wurde, welche in prokaryontischen Signaltransduktionsproteinen weit verbreitet vorkommt. Die Struktur dieser HAMP Domäne sollte aufgeklärt werden, was durch NMR-Spektroskopie erstmalig bei einer HAMP-Domäne gelang. Die HAMP Domäne lag in einer coiled coil Konformation vor, die bisher noch nie bei einem nativen Protein beobachtet wurde: complementary x-da Konformation. Aus dieser Besonderheit konnte ein molekularer Signaltransduktionsmechanismus für prokaryontische Transmembranrezeptoren abgeleitet werden. Der Mechanismus basiert auf der Zahnradartigen Drehung von alpha-Helices. Ein weiterer Aspekt in diesem Schwerpunkt war, nach möglichen Liganden für die extraplasmatische Domäne von Af1503 zu suchen. Als Ergebnis wurde Calcium als potentieller Ligand identifiziert und die Interaktion wurde mit Kalorimetrie charakterisiert. Abschließend wurde eine mögliche funktionelle Interaktion der Proteine des Operons und eine Funktion des Operons für das Archaeum A. fulgidus vorgeschlagen und diskutiert.In three parts the evolution of double psi beta-barrel proteins and aspartic proteases was analyzed. The first part was about the folds double psi beta-barrel to which representatives of N-terminal domains of AAA-ATPases like Vat or Cdc48 belong to, and acid proteases like the aspartic proteases pepsin and HIV-1 protease. Even though these folds have been recognized and described as structurally very similar they are regarded so far to be analogous with convergent development. These assumptions were based on the missing sequence similarity and differences in the connections of their beta-strands. This work suggests a common ancestry based on newly discovered functional similarities. Chaperone assays have been done for that which revealed the prevention of protein aggregation as a common trait. This supplies one more example in which folds changed in the course of divergent evolution. One additional aspect in this part was to examine if the domains of the different folds are interchangeable within their domain context. The second part dealt with the folds double psi bet-barrel and swapped hairpin-barrel. Homology of those has been suspected already due to characteristic structural similarities. However, a low sequence similarity could not support this notion clearly. This homology would be one more example of fold changes during evolution but a evolutionary mechanism had been missing. The structures of the proteins PhS018 from Pyrococcus horikoshii and Af1504 from Archaeoglobus fulgidus were the subject of this work. A new fold has been discovered by solving the solution structure of PhS018: the RIFT-barrels. This fold is intermediate between double psi bet-barrel and swapped hairpin-barrel and provides a plausible evolutionary mechanism for the development of these folds. Moreover, a structurally highly conserved beta/alpha/beta-element could be identified with which an evolutionary scenario from antecedent peptides made of supersecondary structure elements could be proposed. Besides PhS018, the potential double psi beta-barrel protein Af1504 was characterized biochemically. The third part dealt with the function of an operon from A. fulgidus. The protein Af1504 is encoded in it together with the proteins Af1502, Af1503 and Af1505. A HAMP-domain had already been predicted for the transmembrane protein Af1503, which are abundant domains in prokaryotic signal transduction proteins. As the first structure of a HAMP domain ever, the solution structure of the HAMP-domain from Af1503 was solved by NMR spectroscopy. The HAMP domain shows a coiled coil conformation that had never been observed before in a native protein: complementary x-da conformation. Out of this peculiarity, a molecular signal transduction mechanism could be suggested for prokaryotic transmembrane receptors. This mechanism bases on the cogwheel-like rotation of alpha-helices. Another aspect in this part was the search for potential ligands of Af1503. Calcium was suggested to be a ligand and its interaction with Af1503 was investigated and characterized by calorimetry. Finally, a functional interaction of the proteins of this operon and a function of this operon for the archaeon A. fulgidus has been proposed and discussed

A soluble mutant of the transmembrane receptor Af1503 features strong changes in coiled-coil periodicity

Structures of full-length, membrane-bound proteins are essential for understanding transmembrane signaling mechanisms. However, in prokaryotic receptors no such structure has been reported, despite active research for many years. Here we present results of an alternative strategy, whereby a transmembrane receptor is made soluble by selective mutations to the membrane-spanning region, chosen by analysis of helix geometry in the transmembrane regions of chemotaxis receptors. We thus converted the receptor Af1503 from Archaeoglobus fulgidus to a soluble form by deleting transmembrane helix 1 and mutating the surface residues of transmembrane helix 2 to hydrophilic amino acids. Crystallization of this protein resulted in the structure of a tetrameric proteolytic fragment representing the modified transmembrane helices plus the cytoplasmic HAMP domain, a ubiquitous domain of prokaryotic signal transducers. The protein forms a tetramer via native parallel dimerization of the HAMP domain and non-native antiparallel dimerization of the modified transmembrane helices. The latter results in a four-helical coiled coil, characterized by unusually large changes in helix periodicity. The structure offers the first view of the junction between the transmembrane region and HAMP and explains the conservation of a key sequence motif in HAMP domains