Surface contamination of dental implants assessed by gene expression analysis in a whole-blood in vitro assay: a preliminary study.

AIM: We aimed at evaluating pyrogen contamination of dental implants made of titanium and zirconia by using gene expression analysis in a whole-blood in vitro assay. MATERIAL AND METHODS: Titanium and zirconia implants (five each) were incubated in human whole blood. Samples were assayed for gene expression levels of toll-like receptor 4 (TLR4), TLR9, interleukin (IL)-1ß, nuclear factor 'kappa-light-chain-enhancer' of activated B-cells (NF-kB), tumour necrosis factor (TNF)-α, and Fas-associated protein with death domain (FADD) as indicators of surface contamination resulting in lipopolysaccharides (LPS)-stimulated TLR- or TNF-mediated immune responses. Gene expression was assayed using real-time quantitative polymerase chain reaction (RT-qPCR). Non-stimulated blood from the same donor served as a negative control, and blood stimulated with LPS served as a positive control. After dry-heat treatment with dry heat, all implants were re-analysed as described above. RESULTS: Both implant systems contained surface contaminants evoking a pro-inflammatory response similar to that induced by LPS. After dry-heat treatment, gene expression was significantly decreased to levels similar to those of negative control samples. CONCLUSIONS: The results demonstrated LPS-like surface-bound contaminants in both tested implant systems. Depyrogenation with dry heat seems to be an effective means of reducing such contamination in dental implants.

[Effect on elimination of pyrogen and effectual components of Panax notoginseng saponins by ultrafiltration membranes of different cut-off molecular weight and different materials].

OBJECTIVE: To study the elimination effect of bacterial endotoxins and the transmittance of Panax notoginseng saponins by ultrafiltration membranes of different cut-off molecular weight and different materials. METHODS: The kinetic-turbidimetric method was used to determine the content of bacterial endotoxins in Panax notoginseng saponins solution before and after using the ultrafiltration. The change of the contents of active components was examined by HPLC,using notoginsennoside R1, ginsennoside Rg1, ginsennoside Rb1 and ginsennoside Rd as the mark components. RESULTS: The removal rate of bacterial endotoxin fell along with the increasing of membrane aperture. The removal rate was 20. 69% by ultrafiltration membranes of 100 KDa with polysulfone material,less than those of other ultrafiltration membranes with polysulfone material. But the removal rate of bacterial endotoxin by E membranes of blend materials was higher than those of other ultrafiltration membranes with polysulfone material. The contents of active components filtered by E membranes of blend materials was more than that of ultrafiltration membranes of 100 KDa with polysulfone material. CONCLUSION: The applicability of ultrafiltration membranes of large cut-off molecular weight and blend materials of effectual component in Panax notoginseng saponins and elimination of pyrogen is good.

C-reactive protein in patients on chronic hemodialysis with different techniques and different membranes.

In hemodialysis patients, C-reactive protein (CRP), an acute-phase reactant, is a sensitive and independent marker of malnutrition, anemia, and cardiovascular mortality. The aim of the present study was to evaluate CRP levels in plasma samples from long-term hemodialysis patients on different extracorporeal modalities and dialyzed with different membranes, at baseline and after 6 months. Two hundred and forty-seven patients were recruited in eight hospital-based centers. All patients had been on their dialytic modality for at least 3 months and were prospectively followed in their initial dialytic modality for 6 months. Patients were treated with conventional bicarbonate dialysis (N = 127) or hemodiafiltration (N = 120). Patients treated with conventional bicarbonate dialysis were dialyzed with different membranes: Cuprophane (N = 51), low-flux cellulose modified membrane (N = 37) and synthetic membranes (N = 39). Hemodiafiltration was performed in post-dilution mode with polysulfone (N = 66) and polyacrylonitrile (N = 54) membranes. Analysis of baseline CRP values in the clinically stable patients showed that an unexpectedly high proportion (47%) of the patients had CRP values higher than 5 mg/l (upper limit in normal subjects). The mean +/- S.D. CRP values were significantly higher (P < 0.05) in hemodiafiltration with infusion volumes < 10 l per session (14.6+/-3.1 mg/l) than in standard hemodialysis (5.1 +/- 2.1 mg/l) and hemodiafiltration with infusion volumes > 20 l per session (4.9 +/- 2.1 mg/l). These values did not significantly change after 6 months of follow-up. Concerning the membranes, the highest levels of CRP were observed in patients dialyzed with Cuprophane with a significant increase from 15.1 +/- 3.6 to 21.2 +/- 3.1 mg/l after 6 months (P < 0.05); a significant reduction of CRP levels was observed in patients dialyzed with polysulfone in the same follow-up period (from 13.5 +/- 2.9 to 8.1 +/- 2.4 mg/l; P < 0.05). The CRP increase following low volume HDF can be related to back-filtration of bacterial derived contaminants.; moreover, an important effect on CRP of the hemodialysis membrane is observed and new synthetic membranes can be used to decrease these levels.

Immunoadjuvants enhance the febrile responses of rats to endogenous pyrogen.

The febrile responses of male Sprague-Dawley rats to a semipurified endogenous pyrogen produced from human monocytes were characterized by establishing fever dose-response curves. The animals were then injected intravenously with a number of substances that possessed the common properties of stimulating the phagocytic activity of the cells of the reticuloendothelial system and of acting as immunoadjuvants. The substances used were zymosan, lipopolysaccharide endotoxin, and muramyl dipeptide. Three days after any of these immunoadjuvants were injected, the fever sensitivity of the rats was remeasured. In each case, the slope of the fever dose-response curve tripled, and in some instances the response threshold for fever response was reduced by factors of three to eight. Furthermore, the maximum increase in body temperature produced by the endogenous pyrogen was more than doubled after immunoadjuvant treatment. By contrast latex beads, which are also phagocytized by the cells of the reticuloendothelial system but do not subsequently increase their phagocytic index nor do they enhance immune responses, had no effect on the fever sensitivity of rats in response to endogenous pyrogen. In the light of these findings, it is suggested that the febrile responses of rats to endogenous pyrogen are mediated in some manner by cells that possess some of the properties of reticuloendothelial cells. The location of these putative cells must be close to the circulation, because the immunoadjuvants used in this study were, for the most part, large molecular weight molecules that could not cross the blood-brain barrier easily.

Ultrafiltration and endotoxin removal from dialysis fluids.

Biocompatibility in hemodialysis is now regarded as a multifactorial problem and dialysate represents a main risk. Pyrogenic fractions mostly coming from gram-negative bacteria easily pass through dialysis membrane, either by backdiffusion or by backfiltration, and induce blood cell activation. To demonstrate the long-term efficiency of a 2 m2 polyamide ultrafilter in producing a pyrogen free solution, we used an experimental circuit ultrafiltering for 240 hours (500 ml/min) a bicarbonate dialysate contaminated (5 to 48 EU/ml) by a Pseudomonas aeruginosa filtrate. The efficiency was monitored by LAL-test and IL-1 PBMC so to detect not only lipid A containing endotoxins but also other cytokines inducing bacterial fractions. At the post-ultrafilter sampling port the LAL-test was < 0.005 to 0.034 EU/ml; IL-1 PBMC was below the detection limit (20 pg/ml) being 27 to 63 pg/ml at the pre-ultrafilter level. Polyamide ultrafiltration represents an efficient system to obtain an endotoxin-free dialysate and a single filter works up to 240 hours.

Pyrogen retention by the Asahi APS-650 polysulfone dialyzer during in vitro dialysis with whole human donor blood.

The purpose of this study was to test the pyrogen permeability of the new Asahi polysulfone APS 650 (APS) dialyzer membrane with a high permeability for middle molecules (up to 40 kDa) in comparison with the high-flux Fresenius polysulfone F60S (F60S) membrane. Dialyzers were tested in parallel in vitro dialysis experiments with whole human donor blood in the blood compartment and contaminated bicarbonate dialysate in the dialysate compartment. Dialysate was contaminated by a filtrate (0.45 microm) of a Pseudomonas aeruginosa culture in bicarbonate dialysate. The production of interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha) in whole blood samples taken from the in vitro dialysis system was used to detect the passage of cytokine inducing bacterial substances derived from P. aeruginosa across the two highflux polysulfone membranes. Compared with a sterile control period at the beginning of each experiment (n = 5), the TNFalpha inducing activity in the dialysate increased from (mean +/- SEM) 42 +/- 12 pg/ml to 1,288 +/- 356 pg/ml with F60S dialyzers and from 37 +/- 10 pg/ml to 928 +/- 249 pg/ml with APS dialyzers 30 minutes after the dialysate was contaminated. The IL-1beta inducing activity in the dialysate increased similarly. In the presence of this significant contamination in the dialysate, whole blood circulating in the blood compartments for 60 minutes was not stimulated to produce increased amounts of TNFalpha or IL-1beta with neither of the two tested membranes. We conclude that F60S and APS membranes are equal in their ability to prevent the passage of cytokine inducing bacterial substances from highly contaminated dialysate into the patients' blood during hemodialysis.

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.

Preparative separation of pyrogens from proteins by isoelectric focusing using a multicompartment electrolyser with Immobiline membranes.

Due to the nature of lipopolysaccharide endotoxin structures of bacterial pyrogens, their removal from solutions containing therapeutic proteins is often a problem in the pharmaceutical industry. In this report we describe the application of electromotive force to dislodge lipopolysaccharide endotoxins from proteins. This was performed by employing a multicompartment electrolyzer fitted with Immobiline membranes of specified pIs. A thousand-fold reduction of endotoxin could be achieved in the model test system described. This contribution describes the use of a new recycling isoelectric focusing approach without the use of carrier ampholytes.

Purification of murine IgG1 on group specific affinity sorbents.

Murine IgG1 a CD4-antibody, was produced from the cell line MAX 16 H 5. DEAE-Sepharose CL-6B and group specific sorbents based on different ligands, such as protein A-Sepharose, AvidChrom, Thiophilic Resin (T-Resin) Fractogel EMD TA 650S, Sepharose 4B-immobilized metal chelate and histidine sorbents, were compared in terms of their suitability for the large-scale purification of this IgG. Evaluation criteria were selectivity for IgG1 protein capacity and recovery, stability under cleaning-in-place (CIP) conditions and the rate of IgG1 adsorption. Both thiophilic gels performed best under these conditions, with Fractogel EMD TA 650S demonstrating slightly higher contamination with other proteins compared to the T-Resin based on 6% beaded Agarose. Higher purities were obtained with these gels compared to conventional purification techniques employing a combination of ion exchange and hydrophobic interaction chromatography. Favorable adsorption kinetics were found on DEAE-Sepharose with the effective diffusivity in the pores of the sorbent being five-times accelerated compared to the diffusion coefficient of IgG1 in solution. Multilayer adsorption of the IgG1 was experienced from adsorption isotherms on DEAE-Sepharose and Fractogel EMD TA 650S. This was linked to an early breakthrough of this protein during frontal chromatography. Co-purification of pyrogens between two-(Thiophilic Resin) and five-times (DEAE-Sepharose) of the original level was significant with all sorbents except protein A-Sepharose and AvidChrom. Thus an additional chromatographic step is required in case of pyrogen contamination of the cell culture homogenate.