RESUMEN
We analyse the leucocyte and endothelial cell response to polybromostyrene-polystyrene (PS/PBrS) and the poly-n-butylmethacrylate-polystyrene (PnBMA/PS) systems, both in flat form or nanostructured surfaces consisting of nanohills with increasing hill height (13-95nm). Experiments were carried out first with blood leucocytes alone, endothelial cells (of three different types) alone, and finally, using blood cells and endothelized nanosurfaces. Blocking monoclonal antibodies specific for CD11, CD29, CD31, CD54, CD166 were used to analyse whether and to what extent adhesion molecules could be involved in the adherence of both blood leucocytes and endothelial cells to different nanosurfaces. Expression of CD29 (beta-1 integrin), CD54 (ICAM-1) and CD166 (ALCAM) on blood leucocytes was dependent on the hill height, being most prominent with 13nm (PS/PBrS) and 45nm hill (PnBMA/PS) nanosurfaces. Adherence of a human microvascular endothelial cell line and umbilical primary endothelial cells was also related to hill height, being most prominent with 13nm hill height. An indirect correlation was observed between the extent of endothelization and the degree of leucocyte adherence. In cases of low to medium extent of endothelization, the adherence of monocytes and granulocytes was mediated by the expression of CD166, CD29 and CD11a (alpha-L integrin), CD29, CD31 (PECAM-1), respectively. Scanning electron microscopy studies showed the predominant emission of pseudopodia at the holes of the surfaces and the focal contacts with the nanosurfaces. Our studies emphasize the relevance of testing functional properties in co-culture experiments in the development and optimization of nanosurfaces for biomedical application.
Asunto(s)
Endotelio Vascular/citología , Endotelio Vascular/fisiología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/fisiología , Nanotecnología/métodos , Poliestirenos , Adhesión Celular/fisiología , Células Cultivadas , Cristalización/métodos , Endotelio Vascular/ultraestructura , Humanos , Leucocitos Mononucleares/ultraestructura , Ensayo de Materiales , Venas Umbilicales/citología , Venas Umbilicales/fisiologíaRESUMEN
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.
Asunto(s)
Endotoxinas/aislamiento & purificación , Soluciones para Hemodiálisis , Membranas Artificiales , Diálisis Renal , Células Cultivadas , Contaminación de Medicamentos , Humanos , Interleucina-1/biosíntesis , Leucocitos Mononucleares/metabolismo , Peso Molecular , Pirógenos/aislamiento & purificación , UltrafiltraciónRESUMEN
A complex array of inflammatory mediators are generated as a consequence of blood contact with hemodialysis (HD) membranes. Beside complement activation, other mediators are involved in cell activation, and are thought possibly to be responsible for early and long-term multiple changes in immunity infection, hypercatabolism, beta 2-microglobulin generation and hemostatic mechanisms. Previous studies from our laboratories have established platelet activating factor (PAF) as one of the mediators generated by complement-dependent or independent mechanisms of cell interaction with hemodialysis membranes. Recent studies on the production of PAF from endotoxin-primed polymorphonuclear neutrophils in a closed miniaturized circuit, and on the effect of PAF in mediating endotoxin- and cytokine-induced leukocyte adherence to HD membranes, highlight so far undescribed new roles of this mediator in biocompatibility.
Asunto(s)
Factor de Activación Plaquetaria/fisiología , Diálisis Renal/efectos adversos , Fenómenos Fisiológicos Sanguíneos , Endotoxinas/efectos adversos , Humanos , Riñones Artificiales/efectos adversos , Leucocitos/fisiología , Membranas Artificiales , Neutrófilos/fisiologíaRESUMEN
An in vitro system composed of a plasma separation membrane coupled with natural (charcoal) or synthetic (Amberlite, Amberchrome) types of sorbents was evaluated for the simultaneous removal of proinflammatory cytokines (TNF-alpha, IL-1 beta and IL-8) and cytokine antagonists [interleukin (IL)-1 receptor antagonist (IL-1Ra), soluble tumor necrosis factor-alpha (TNF-alpha) receptor I and II (sTNFR I and II)] in whole blood spiked with bacterial lipopolysaccharide (LPS). These studies showed that plasma filtration rather than ultrafiltration significantly increased the clearance of all cytokines, particularly TNF-alpha, and the synthetic (Amberlite-type of resin) but not natural (uncoated charcoal) membrane could extensively absorb almost 100% of plasma filtered IL-Ra, IL-1 beta and IL-8, but only 40% of TNF-alpha. Other synthetic (Amberchrome) membranes could also effectively (80%) remove TNF-alpha. In the complex scenario of sepsis, the simultaneous removal of excess proinflammatory and/or immunomodulatory mediators may play a role in reducing the hemodynamic alterations, thus resulting in enhanced patient survival. Whether this occurs in the human setting awaits the results of an ongoing clinical investigation.
Asunto(s)
Hemofiltración/métodos , Adsorción , Carbón Orgánico , Cuidados Críticos , Citocinas/sangre , Citocinas/aislamiento & purificación , Hemofiltración/instrumentación , Humanos , Mediadores de Inflamación/sangre , Mediadores de Inflamación/aislamiento & purificación , Plasma , Resinas Sintéticas , Sepsis/sangre , Sepsis/terapiaRESUMEN
Platelet-activating factor is a recognized mediator of anaphylaxis and bioincompatibility. Here, the mechanisms and the kinetics of the production of platelet-activating factor were studied in vivo during high-flux hemodialysis and in vitro in a recirculation model with polyacrylonitrile membranes, the AN-69 and the more recent SPAN, where the Na-metallilsulfonate group is partially substituted with the less polar methacrylate group. In in vivo studies, eleven patients were studied in cross over. Patients were randomly allocated to the AN-69 (5 patients) and to the SPAN membrane (6 patients) for two weeks. Measurements were made in the second week of use. After completion of the second week, the patients were switched to the other membrane for a further two weeks. Samples for leukocyte and platelet counts, PAF in whole blood or bound to platelets, the C3a des Arg and the C5b-C9 membrane attack complex as well as samples for clearances of urea, creatinine and phosphates were taken at different time intervals during treatment. PAF was detected by biological assay after methanol extraction of whole blood or of platelet pellets obtained by sequential centrifugation. C3a des Arg and the C5b-C9 fraction were detected by commercially available immunoassays. Results were analyzed by Minitab statistical package. PAF was detectable only during treatment with AN-69 but not with SPAN 1 min after start of the extracorporeal circulation in both whole blood (4.5 +/- 2.7 ng/ml) and on platelet surface (4.1 +/- 1.2 ng/ml). No statistical significant differences were observed between AN-69 and SPAN with regard to leukocyte and platelet counts, plasma C3a des Arg and C5b-C9 levels. The structure modification did not alter functional performances as indicated by the lack of statistically significant differences in clearance values between the two membranes. In in vitro experiments performed with normal washed and whole blood recirculated in a closed circuit demonstrated the presence of a plasma-dependent, complement-independent mechanisms responsible for the triggering of PAF synthesis and release with AN-69 but not SPAN membrane. PAF was extractable from the inner and outer side of both polyacrylonitrile membranes (AN-69: inner, 4.9 +/- 0.5 ng/ml; outer, 0.1 +/- 0.05 ng/ml; SPAN: inner, 5.5 +/- 0.6 ng/ml, outer: 3.3 +/- 0.7 ng/ml, SPAN vs. p < 0.001), suggesting that absorption may be relevant with both membranes.
Asunto(s)
Resinas Acrílicas/metabolismo , Membranas Artificiales , Factor de Activación Plaquetaria/biosíntesis , Diálisis Renal/instrumentación , Insuficiencia Renal/terapia , Anciano , Materiales Biocompatibles , Complemento C3/metabolismo , Complemento C5/metabolismo , Complemento C5b , Complemento C9/metabolismo , Femenino , Humanos , Técnicas para Inmunoenzimas , Recuento de Leucocitos , Espectroscopía de Resonancia Magnética , Masculino , Persona de Mediana Edad , Recuento de Plaquetas , Insuficiencia Renal/inmunologíaRESUMEN
Lipopolysaccharide (LPS) from gram negative bacteria has been shown to prime human polymorphonuclear neutrophil (PMN) production of platelet activating factor (PAF). PAF is a lipid mediator of inflammation and endotoxic shock and is also involved in leukocyte activation occurring during hemodialysis; PAF induces leukopenia, degranulation of lysosomal granules, and adherence to hemodialysis membranes. Transmembrane passage of LPS has also been shown to occur. To evaluate the relevance of transmembrane passage of LPS on the priming of PMN derived production of PAF, we designed in vitro studies using an experimental circuit equipped with different membranes (Cuprophan, polysulfone, polymethylmethacrylate, polyamide) and recirculation of purified human PMNs. At different time intervals, PMNs were stimulated with FMLP (10 microM) after back-filtration of sterile and LPS contaminated dialysate. The results of these studies suggest that priming of PMN derived production of PAF was related to the percent of backfiltered LPS, and they emphasize the need for careful assessment of microbiologic quality to improve biocompatibility.
Asunto(s)
Endotoxinas/inmunología , Riñones Artificiales , Lipopolisacáridos/inmunología , Membranas Artificiales , Neutrófilos/inmunología , Factor de Activación Plaquetaria/biosíntesis , Electroforesis en Gel de Poliacrilamida , Contaminación de Equipos , Humanos , Modelos CardiovascularesRESUMEN
To reduce the level of contamination by bacterial products, ultrafiltration systems have been introduced and validated for their capacity to block the passage of bacterial components reactive to the limulus amoebocyte lysate (LAL) test. In this study, the absorptive capacity of polysulfone membranes undergoing disinfection cycles with free chlorine and peracetic acid were evaluated at various concentrations and contact times. The results of this study implicate a relevant physicochemical derangement of the polysulfone membranes treated with sodium hypochlorite but not with peracetic acid, diluted peracetic acid (Dialox) or Amuchina. The implications for the practical use of ultrafilters are discussed.
Asunto(s)
Desinfectantes , Prueba de Limulus , Membranas Artificiales , Polímeros , Diálisis Renal , Sulfonas , UltrafiltraciónRESUMEN
Regenerated cellulosic membranes (CU) induced the aggregation of plasma-free human neutrophils when recirculated in a dynamic model of dialysis without the patient on the circuit. Neutrophil aggregation was linked to the production of PAF by these cells. In the absence of detectable PAF production, no neutrophil aggregation occurred, as observed during recirculation with polymethylmethacrylate (PMMA) membranes. With polycarbonate (PC), PAF production and aggregation of neutrophils were both almost half the values with CU. PAF production was studied in ten hemodialysis (HD) patients tested twice with CU and once with PC and PMMA membranes. PAF was extracted in the venous blood during filling of the dialyser for 9/20 of patients with CU (3.1 +/- 2.9 ng/ml, mean +/- 1 S.D.) a membrane that induced marked leukopenia (greater than 50% of basal values at 15 min), C3a des Arg generation (greater than 500% at 5 min), and plasma levels of the elastase-alpha 1-proteinase inhibitor complex (greater than 500% at the end of HD). Membranes such as PC and PMMA showing intermediate or low potential to induce leukopenia and C3a des Arg generation, respectively, did not trigger the production and release of PAF in detectable amounts at any interval. However, with PMMA, plasma neutrophil elastase was significantly higher than baseline at the end of dialysis. These levels were not significantly different (p less than 0.05) from those observed with CU and PC membranes.
Asunto(s)
Membranas Artificiales , Neutrófilos/metabolismo , Factor de Activación Plaquetaria/metabolismo , Diálisis Renal , Agregación Celular , Celulosa , Femenino , Humanos , Masculino , Metilmetacrilatos , Persona de Mediana Edad , Cemento de PolicarboxilatoRESUMEN
Polymethylmethacrylate (PMMA) membranes with different net electric charges and percentage water contents (anionic 71%, neutral 70%, cationic 75%) were evaluated for their ability to stimulate plasma-free human polymorphonuclear neutrophils (PMN), and compared for potency to cuprophan (Cu), already described as being a potent trigger of PMN. The release of lysozyme, beta-glucuronidase, lactic dehydrogenase (LDH), and the generation of a platelet aggregating activity were studied in the supernatants from plasma-free human PMN incubated with different membranes. The PMN intracellular content of neutrophil cationic proteins (NCP), elastase, and cathepsin G were also studied by immunofluorescence using specific antisera on smears of PMN before and after incubation with each membrane. Only cationic, but not anionic or neutral PMMA induced a marked release of lysozyme (range 20-25% of the sonicated control, assumed as 100%), and beta-glucuronidase (40-43%), and marked depletion of the intracellular content of NCP, elastase, and cathepsin G, suggesting a degranulation process. Platelet aggregating activity was generated and referred to the release of platelet activating factor (PAF) only in the supernatants from PMN incubated with cationic, but not with anionic, or neutral PMMA membranes. These results indicate that modification of the net electric charge can per se turn PMMA, commonly recognized as inert, into a material with marked PMN activating effects, comparable to those of Cu, a highly reactive polymer.
Asunto(s)
Cationes/metabolismo , Membranas Artificiales , Metilmetacrilatos/metabolismo , Neutrófilos/metabolismo , Diálisis Renal , Celulosa/análogos & derivados , Celulosa/metabolismo , Glucuronidasa/metabolismo , Humanos , Muramidasa/metabolismo , Agregación PlaquetariaRESUMEN
BACKGROUND: Based on the increased hydraulic permeability of the new high permeability polyethersulfone membrane, DIAPES HF-800, we investigated the kinetics and handling of albumin in high volume on-line hemodiafiltration (HDF). METHODS: Seven patients on predilutional HDF were studied in two consecutive sessions. Blood flow rate and transmembrane pressure were continuously monitored. Spent dialysate was spilled at 20 ml/h every hour. Albumin was measured in blood and dialysate by immunonephelometry. Albumin and proteins adsorbed onto the dialyzer membrane were eluted after treatment with Triton X. Ultrafiltrates collected at 1 and 2 hours of treatment were pooled from different patients and incubated for 24 hours at 37 degrees C with bovine serum albumin (BSA). Total sulphydryl groups were evaluated using Ellmann's reagent [5, 5'-dithio-bis(2-nitrobenzoic acid)]. RESULTS: In all 7 patients, the total loss of albumin was 3.99 +/- 1.81 g, ranging between 1.09 and 6.82 g/session. Most albumin loss occurred in the first 60 min of pre-dilutional hemodiafiltration (1.92+0.83 g). There was no correlation between transmembrane pressure, urea clearance and the loss of albumin. Plasma water urea clearance values were stable over the treatment (234 +/- 14.3 ml/min). Plasma albumin concentration did not decrease during HDF sessions. Albumin adsorbed onto the dialyzers was 0.7 +/- 1.6 mg but the total amount of adsorbed proteins was much higher (130 + 90 mg). In addition, the ultrafiltrate collected during HDF sessions was able to induce oxidation of bovine serum albumin as measured by total protein sulfhydryl groups: bovine serum albumin incubated in the presence of ultrafiltrate collected at 1 hour had a sulfhydryl loss of 56.3 +/- 5.7% (p < 0.0001 vs control), and bovine serum albumin incubated with ultrafiltrate collected at 2 hours had a loss of 67.5 +/- 3.8% (p < 0.003 vs control). CONCLUSION: The present study shows the high inter- and intra-patient variability of transmembrane passage of albumin in chronically uremic patients undergoing pre-dilutional HDF. Factors involved do not seem to be correlated to transmembrane pressure but rather to an interaction with the polymer surface. Albumin adsorption was minimal and was significantly lower than that of other plasma proteins. Albumin loss during HDF seemed to have no acute impact on plasma albumin. In addition, we demonstrated the presence of prooxidative compounds able to oxidize albumin, of which extracorporeal removal by HDF procedure could be beneficial for HD patients.
Asunto(s)
Hemodiafiltración , Albúmina Sérica/análisis , Adsorción , Anciano , Albúminas/análisis , Soluciones para Diálisis/química , Femenino , Humanos , Técnicas In Vitro , Masculino , Membranas Artificiales , Persona de Mediana Edad , Oxidación-Reducción , Permeabilidad , Polímeros , Albúmina Sérica Bovina/análisis , Compuestos de Sulfhidrilo/análisis , Sulfonas , Urea/metabolismoRESUMEN
Ethylene oxide (ETO) is presently the most commonly used sterilization method for medical devices. Although alternative sterilization modes such as steam sterilization have been suggested, the effect of steam on dialysis-induced cytokine release is unknown. We enrolled 9 patients on chronic hemodialysis and evaluated at different intervals IL-1beta production while treated with ETO (NC 1785-Bellco) and steam sterilized NC 1785S-Bellco) Synthetically Modified Cellulose (SMC). A basal test during treatment with NC 1785 was performed (A); the same test was set up 4 weeks after treatment with NC 1785S (B) and, lastly, 4 weeks after returning to NC 1785 (C). Peripheral blood mononuclear cells (PBMC) were purified before and after the dialysis session, were isolated on a Ficoll/Hypaque gradient and incubated for 24 h. Spontaneous IL-1beta release was evaluated in the supernatant and in the lysate. In A, IL-1beta levels were (in pg/ml/10(6) cells, in supematant and lysate, respectively): 5.8 +/- 4.8 and 7.6+/-5.2 in pre-HD and 4.68 +/- 3.6 and 9.7 +/- 6.65 in post-HD. These levels showed a clear reduction in B: 2.5 +/- 2.2 and 4.4 +/- 3.1 in pre-HD, and 4.35+/- 6.6 and 7.52 +/- 7.22 in post-HD. In the C test, 4 weeks after the return to the ETO membrane, IL-1beta levels remained unchanged: 2.9 +/- 1.8 and 4.5 +/- 3.1 in pre-HD; and 2.6 +/- 3 and 5.7 +/- 6.6 in post-HD. Statistical analysis showed significant changes in the pre-HD levels both in supematant (p < 0.04) and in lysate (p < 0.04). Steam sterilization of SMC induced a lower spontaneous IL-1beta release, but this effect was not statistically significant due to the large inter-individual variation. Hence, contrary to claims of better biocompatibility, steam sterilization does not result in a reduced production of pro-inflammatory IL-1beta.
Asunto(s)
Interleucina-1/análisis , Diálisis Renal , Vapor , Esterilización/métodos , Materiales Biocompatibles , Células Cultivadas , Celulosa/química , Desinfectantes/uso terapéutico , Escherichia coli/química , Óxido de Etileno/uso terapéutico , Humanos , Prueba de Limulus , Membranas Artificiales , Persona de Mediana Edad , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Regenerated cellulosic membranes are held as bioincompatible due to their high complement - and leukopenia - inducing properties. Adherence of polymorphonuclear neutrophils and monocyte purified from normal human blood to the three membranes were evaluated in an in vitro recirculation circuit in the presence or absence of fresh, autologous plasma after recirculation in an in vitro circuit using minimodules with each of the three membranes. In in vivo studies, 9 patients were treated with conventional haemodialysis for 2 weeks with each membrane and 1 week for wash-out using haemodialysers with the following surface: 1.95 m2 for benzyl-cellulose, 1.8 m2 for acetate-cellulose and low-flux polysulfone. Measurement of leukopenia, plasma C3a des Arg and elastase-alpha1 proteinase inhibitor complex levels as well as urea, creatinine, phosphate and uric acid clearances was performed. Plasma-free neutrophils adhered maximally to acetate-cellulose (65% remaining in the circulation), while there was no significant difference between low-flux polysulfone and benzyl-cellulose (80% circulating neutrophils, at 15 min, p<0.001 vs acetate cellulose). In the presence of fresh plasma, as source of complement, the differences between acetate cellulose vs polysulfone and benzyl-cellulose were even more evident, suggesting the role of complement-activated products in neutrophil adherence. A similar trend was observed for monocyte adherence with the three membranes in the absence or presence of plasma. In vivo studies showed that the nadir of leukopenia was at 15 and 30 min with acetate-cellulose (79%) and benzyl-cellulose (50%) (p<0.05 acetate- vs benzyl-cellulose) and at 15 min with polysulfone (24%) (p<0.01 vs acetate- and benzyl-cellulose). Plasma C3a des Arg levels arose to 2037 +/- 120 ng/ml, 1216 + 434 ng/ml and 46 +/- 55 ng/ml with acetate-, benzyl-cellulose and polysulfone, respectively. No pre- vs post-dialysis increase in the intracellular content of TNF-alpha was detected with any of three membranes. Clearance values of urea, creatinine and uric acid were superimposable for all the three membranes. However, benzyl cellulose had a significantly higher clearance for phosphorus (normalized for surface area) (p<0.01 vs acetate-cellulose, 0.001 vs polysulfone). These results implicate that synthetic modification of the cellulose polymer as for the benzyl-cellulose significantly reduces the in vitro adherence, delays the in vivo activation of "classic" biocompatibility parameters and notably improves the removal of inorganic phosphorus.
Asunto(s)
Materiales Biocompatibles , Celulosa/análogos & derivados , Membranas Artificiales , Diálisis Renal , Anciano , Anciano de 80 o más Años , Anafilatoxinas/análisis , Recuento de Células Sanguíneas , Adhesión Celular , Complemento C3a/análogos & derivados , Complemento C3a/análisis , Humanos , Técnicas In Vitro , Fallo Renal Crónico/terapia , Leucopenia/etiología , Persona de Mediana Edad , Monocitos/fisiología , Neutrófilos/fisiología , Elastasa Pancreática/sangre , Fosfatos/sangre , Polímeros , SulfonasRESUMEN
The Authors review the mechanisms involved in the production of cytokines during substitutive extracorporeal treatment with particular reference to microbial contamination and possibility of backfiltration of bacterial constituents more likely to occur with high permeability membranes. Recent and on-going studies from our laboratory support the contention that patients treated with high permeability membranes may be chronically stimulated. Use of "ultrapure" solution indeed brings about a marked reduction in predialytic plasma levels of tumor necrosis factor (TNF) in regard to what observed when standard solutions are adopted.
Asunto(s)
Hemofiltración/efectos adversos , Diálisis Renal/efectos adversos , Factor de Necrosis Tumoral alfa/biosíntesis , Resinas Acrílicas , Acrilonitrilo/análogos & derivados , Materiales Biocompatibles , Contaminación de Medicamentos , Endotoxinas , Contaminación de Equipos , Soluciones para Hemodiálisis , Hemofiltración/instrumentación , Humanos , Membranas Artificiales , Metilmetacrilatos , Polímeros , Diálisis Renal/instrumentación , Sulfonas , Factor de Necrosis Tumoral alfa/análisisAsunto(s)
Materiales Biocompatibles , Leucocitos/fisiología , Membranas Artificiales , Factor de Activación Plaquetaria/fisiología , Diálisis Renal , Proteínas del Sistema Complemento/fisiología , Humanos , Leucocitos/inmunología , Factor de Activación Plaquetaria/biosíntesis , Diálisis Renal/efectos adversosRESUMEN
A simple immunohistological test has been developed to detect and to quantitate the presence of immune complexes (IC) on the surface of human polymorphonuclear neutrophils (PMN). The interaction between IC and PMN has been evaluated in several human IC diseases. High amounts of immunoglobulins (Ig) and C3 were detected on the PMN surface from these patients. The deposits of Ig and C3 were inversely related to the percentage of membrane-free receptors for Fc and C3.
Asunto(s)
Enfermedades del Complejo Inmune/inmunología , Neutrófilos/inmunología , Fosfatasa Ácida/sangre , Fosfatasa Alcalina/sangre , Humanos , Enfermedades del Complejo Inmune/diagnóstico , L-Lactato Deshidrogenasa/sangre , Lupus Eritematoso Sistémico/inmunología , Fagocitosis , Polietilenglicoles/sangre , Receptores de Antígenos de Linfocitos BRESUMEN
Pyrogen permeability of the new highly permeable synthetic membrane polyethersulfone (DIAPES) was compared to polysulfone in vitro dialysis experiments with heparinized human donor blood in the blood compartment. After sterile dialysis for 5 min, dialysate was contaminated with a culture filtrate of Pseudomonas aeruginosa using high and moderate challenge doses (Limulus assay reactivity 20,000 EU/ml and 50 EU/ml, respectively). Whole blood samples were separated from the blood compartment during the sterile (5 min) and contaminated (60 min) phases of dialysis and incubated for 6 h at 37 degrees C. Blood samples were lysed, and interleukin-1beta and tumor necrosis factor alpha were measured by specific ELISAs. Moderate dialysate contamination (50 EU/ml) did not induce detectable cytokine production in whole blood. High challenge dose (20,000 EU/ml) induced whole blood interleukin-1beta and tumor necrosis factor alpha production in the blood compartment, which was higher with DIAPES than with polysulfone after 30 min. After 60 min, membrane-dependent differences were no longer detectable. Pyrogen concentrations in the dialysate decreased with time indicating adsorption of cytokine-inducing substances to the dialyzer membrane. Pyrogens adsorbed to dialyzer membranes were resuspended during recirculation of sterile phosphate-buffered saline in the dialysate compartment and retained cytokine-inducing activity as seen from whole blood incubation experiments. DIAPES and polysulfone adsorbed pyrogens in the presence of whole blood. Pyrogen adsorption to the membrane polymer and/or to the protein coat on the membrane prevented the passage of pyrogens in the presence of moderately contaminated dialysate. High grade dialysate contamination caused breakthrough of pyrogens into the blood with DIAPES and polysulfone. In order to reduce the risk of a dialysis-dependent inflammatory response, dialysate of high bacteriological quality (ultrapure dialysate) should be mandatory.
Asunto(s)
Membranas Artificiales , Diálisis Renal , Adsorción , Humanos , Interleucina-1/sangre , Permeabilidad , Polímeros , Polimixina B/uso terapéutico , Sulfonas , Factor de Necrosis Tumoral alfa/análisisRESUMEN
Uremic patients undergoing hemodialysis often have increased oxidant stress and accumulation of uremic toxins. Hemodialysis, per se, often can exacerbate oxidant stress and may be inefficient at removing hydrophobic or protein bound toxins. We describe a new hemodialytic method that incorporates liposomes and antioxidants to remove hydrophobic/uremic toxins and minimize free radical mediated damage. In vitro experiments measured advanced oxidation protein products (AOPP), malonaldehyde, reactive carbonyls, and the removal of platelet activating factor (PAF) and bilirubin during extracorporeal circulation with or without liposomes. We observed a significant reduction of oxidation products as well as a significant removal of PAF and bilirubin compared to normal hemodialysis.
Asunto(s)
Liposomas/uso terapéutico , Estrés Oxidativo/fisiología , Diálisis Renal/métodos , Toxinas Biológicas/sangre , Animales , Antioxidantes/uso terapéutico , Ácido Ascórbico/uso terapéutico , Bilirrubina/sangre , Proteínas Sanguíneas/química , Bovinos , Circulación Extracorporea , Depuradores de Radicales Libres/uso terapéutico , Humanos , Malondialdehído/sangre , Oxidación-Reducción , Fosfatidilcolinas/uso terapéutico , Fosfolípidos/uso terapéutico , Factor de Activación Plaquetaria/química , Diálisis Renal/instrumentación , Albúmina Sérica Bovina/química , Uremia/sangre , Uremia/terapia , Vitamina E/uso terapéuticoRESUMEN
On-line hemodiafiltration is a technique that relies on the re-injection of pyrogen-free substitution fluid obtained by cold filtration of dialysate. Therefore, safety of this treatment modality depends on the quality of dialysate and, mainly, on the integrity of the ultrafilter(s) employed. Double-chamber on-line hemodiafiltration is a new technique where re-infusion takes place inside the dialyser by means of dialysate backfiltration. The peculiar geometry of the dialyser allows intra-treatment assessment of its fibre integrity. In this paper, we tested feasibility and safety of this new modality of on-line treatment. The extracorporeal blood and infusate pressure values resulted well inside the safety range. Blood urea clearances and beta(2) removal were consistent with the figures usually found in standard hemodiafiltration. Whole blood production of cytokines was similar when blood was exposed to saline or infusate, both values being comparable to the spontaneous whole blood cytokine release. The on-line dialyser fibre integrity check showed a great sensitivity even for minimal dialyser damage. We conclude that double-chamber on-line hemodiafiltration is a feasible and safe procedure. Our preliminary results encourage the undertaking of multicentre, prospective, randomised studies.