Cuprophane Research Articles

Adsorption of nafamostat mesilate by hemodialysis membranes.

The adsorption of the anticoagulant nafamostat mesilate (FUT-175) by five different hemodialysis membranes was studied in vivo and in vitro. In vivo, FUT-175 was adsorbed strongly by a polyacrylonitrile (AN69) membrane and slightly by another polyacrylonitrile (J-PAN) membrane but not by Cuprophan (CU), hemophan (HE), or polymethylmethacrylate (PMMA) membranes during hemodialysis performed in 4 patients in whom FUT-175 was used as an anticoagulant. Only during hemodialysis using the AN69 membrane did FUT-175 not induce a significant prolongation of celite-activated coagulation time. In vitro studies showed that FUT-175 was adsorbed by the AN69, J-PAN, and PMMA membranes but not by the CU and HE membranes. Methylene blue, a dye that possesses a cationic portion in its chemical structure, stained AN69, J-PAN, and PMMA membranes. Since FUT-175 also possesses a cationic portion, we conclude that FUT-175 is adsorbed by negatively charged membranes via an ionic bond and is unsuited for use as an anticoagulant in hemodialysis using an AN69 membrane because of that membrane's marked capacity to adsorb FUT-175.

The Effect of Hemodialysis and Dialyzer Biocompatibility on Erythrocyte Glutathione-Defense System in Chronic Hemodialysis Patients

Uremic patients, especially those receiving regular hemodialysis (HD) treatment, are at high risk of oxidative damage by noxious free radicals and reactive oxygen species (ROS). The erythrocyte glutathione-defense system (GSH-DS) is one of the major enzymatic means of scavenging and detoxifying ROS. This study aimed to elucidate the effect of HD and dialyzer biocompatibility on erythrocyte GSH-DS in uremic patients on maintenance HD treatment. Twenty-five healthy volunteers and 42 HD patients were enrolled in this study. Blood samples were drawn immediately before and after HD session, and erythrocyte glutathione (GSH) level as well as the activities of the enzymes glucose-6-phosphate dehydrogenase (G6PD), glutathione peroxidase (GSH-Px), glutathione reductase (GSSG-Rd), and glutathione S-transferase (GST) were measured. To evaluate the effect of dialyzer type on the studied parameters the patients were were subdivided into two groups: those who had dialysis with cuprophane (CU) membranes (n=23) and those who received dialysis with the aid of polysulfone (PS) membranes (n=19). The activities of G6PD and GSH-Px as well as GSH level were significantly decreased in HD patients as compared with controls. On the other hand, the activities of GSSG-Rd and GST were significantly elevated among HD patients in comparison with control values. A single HD session, regardless of the type of dialyzer, did not induce any significant effect on any of the measured parameters, although G6PD activity increased significantly after dialysis. CU membrane did not result in any change in GSH or its metabolizing enzymes, while PS dialyzers exerted a minor but significant restoration in GSH-DS. The antioxidant pool, as represented by GSH-DS, is significantly affected by dialyzer type in HD patients being significantly corrected with polysulfone dialyzer.

Biocompatibility Parameters of Different Dialysis Membranes Assessed during Systemic Inflammation

Background: We explored whether biocompatible dialyzer membranes modulate the inflammatory response during blood contact in patients with systemic inflammation. Methods: 15 patients with end-stage renal disease and systemic inflammation (mean serum C-reactive protein 86 ± 4 mg/l) were randomly treated with Cuprophan (CU), polyamide (PA) and vitamin-E coated (VEC) membrane-based dialyzers. Results: Changes in blood pressure, capillary blood oxygen saturation and differential blood counts during the hemodialysis session were not significantly different between the three dialyzers. Baseline blood levels of activated circulating complement (C3a) were more than 100 times above normal, and unlike expected they decreased during hemodialysis treatments (CU: from 7,389 ± 783 to 5,423 ± 761 ng/ml; PA: from 7,379 ± 980 to 5,690 ± 714 ng/ml; VEC: from 7.377 ± 714 to 5,360 ± 1,005 ng/ml; all n.s.). No significant differences between treatments were found with respect to changes in blood concentrations of TNF-α, interleukin-6 and interleukin-1 receptor antagonist as well as ICAM-1 (CU: from 451 ± 41 to 477 ± 41 ng/ml; PA: from 437 ± 42 to 449 ± 40 ng/ml; VEC: from 461 ± 43 to 460 ± 47 ng/ml). Furthermore, generation of reactive oxygen species by mononuclear blood cells was comparable during hemodialysis with the CU, PA and VEC dialyzer. Conclusion: The choice of dialyzer membrane material does not affect most aspects of biocompatibility when patients have significant systemic inflammation. This confounding variable should be taken into account in studies exploring the effects of biocompatible dialyzer membranes.

Apoptosis of human polymorphonuclear neutrophils accelerated by dialysis membranes via the activation of the complement system

Haemodialysis (HD) with bioincompatible cellulosic membranes like Cuprophan (CU) is considered to influence negatively the clinical outcome of acute and chronic renal failure. In this effect, apart from the disturbance of phagocytosis or oxygen species production by leukocytes, increased apoptosis also has been implicated recently. The objective of this study was to study the effect of HD membranes on apoptosis induction in polymorphonuclear neutrophils (PMN). PMN from healthy donors and uraemic patients were isolated and apoptosis was induced by co-incubation with CU, Hemophan or polyamide hollow fibres in the presence of serum from healthy or uraemic humans. Apoptosis was quantified by flow cytometry using Annexin V-FITC and propidium iodide staining and was confirmed by the detection of DNA fragmentation on gel electrophoresis. The deposition of immunoglobulins (Ig) and complement factors on hollow fibres was detected by direct immunofluorescence. Heat inactivation or the depletion of complement components or Ig significantly reduced apoptosis, indicating its dependence on classical complement activation. The detection of IgG on hollow CU fibres and the restored acceleration of apoptosis by the appropriate replenishment of Ig-deficient sera additionally confirmed these findings. Inhibition experiments revealed that caspases were necessary mainly, but not exclusively, for apoptosis to occur after complement activation. Uraemia led to increased PMN apoptosis in the presence of bioincompatible, but not biocompatible, membranes. Our results suggest that the acceleration of PMN apoptosis in the presence of CU is mediated via an antibody-dependent activation of the classical complement pathway mobilizing both caspase-dependent and -independent pathways.

Dialyzer membrane type and reuse practice influence polymorphonuclear leukocyte function in hemodialysis patients

Polymorphonuclear leukocyte (PMNL) production of reactive oxygen species (ROS) has been linked to hemodialysis (HD) associated morbidity. The effect of dialyzer membrane type and reuse on PMNL function has not been clearly defined. The present report is a cross-sectional study undertaken in a cohort of patients undergoing regular HD, at enrollment into the Hemodialysis (HEMO) Study, to study the association between patient and dialysis-related factors and PMNL function. PMNL function was assessed by measuring PMA- and N-formyl methionyl-leucyl-phenylalanine (fMLP) -induced respiratory burst, and phagocytic activity toward Staphylococcus aureus. PMNL from patients dialyzed with polysulphone (PS) or cuprophane (CU) membranes showed higher PMA-induced respiratory burst activity compared with those exposed to substituted cellulose (cellulose acetate, cellulose triacetate, CA/CT) membranes, regardless of dialyzer reuse. The use of bleach as a cleansing agent during reuse was associated with higher PMA-induced PMNL superoxide production, as was the use of renalin when compared to aldehydes. In a subgroup of patients using PS dialyzers, reuse itself was associated with higher fMLP-induced superoxide production. The type of bleach-germicide combination during reuse showed that use of renalin as a germicide was also associated with higher PMNL phagocytosis index. The number of years on HD correlated inversely with PMA-induced PMNL superoxide response. Weaker PMNL response to fMLP was associated with greater comorbidity and poor functional status as quantified by Index of Coexisting Diseases (ICED) and Karnofsky scores, respectively. Our results indicate that dialyzer membrane type and the reuse process influence the oxidative response of PMNL among HD patients. The implications of these observations on clinical morbidity need to be further evaluated in prospective studies.

Induction of Cytokines and Adhesion Molecules in Stable Hemodialysis Patients: Is There an Effect of Membrane Material?

Background/Aims: The aim of this study was to examine the effects of a biocompatible and a nonbiocompatible hemodialysis membrane on the secretion of cytokines and their specific antagonists as well as on the expression of adhesion molecules. Methods: A crossover study using cuprophane (CU) and polysulfone (PS) dialyzers and measurements of serum tumor necrosis factor (TNF)-alpha, soluble TNF receptors, interleukin (IL)-6, IL-6 receptor, IL-2 receptor, IL-1 receptor antagonist and the adhesion molecules intercellular adhesion molecule 1, endothelial leukocyte adhesion molecule and vascular cell adhesion molecule was performed in 19 hemodialysis patients. 44 healthy volunteers served as controls. Results: All of the measured cytokines and cytokine antagonists as well as all adhesion molecules were significantly elevated in hemodialysis patients compared to controls. There was a significant increase in TNF-alpha during a dialysis session with a CU dialyzer, but only a moderate increase using a PS dialyzer. None of the other cytokines and adhesion molecules were changed during a dialysis session. We were also able to show an upregulation of adhesion molecules in dialysis patients. Conclusion: Our study clearly demonstrates that levels of inflammatory cytokines as well as their antagonists and adhesion molecules are elevated in patients on hemodialysis therapy. Baseline values before the start of a dialysis session did not show any differences with regard to the usage of CU or PS dialysis membranes. However, CU dialyzers led to a significantly greater stimulation of TNF-alpha during the dialysis session in comparison to PS membranes, suggesting a higher degree of bioincompatibility.

Gap junction communication between cells aggregated on a cellulose-coated polystyrene: influence of connexin 43 phosphorylation

The appropriate functioning of tissues and organ systems depends on intercellular communication such as gap junctions formed by connexin (Cx) protein channels between adjacent cells. We have previously shown that Swiss 3T3 cells aggregated on hydrophilic cellulose substratum Cuprophan (CU) establish short linear gap junctions composed of Cx 43 in cell surface plaques. This phenomenon seems to depend on the high intracellular cyclic AMP (cAMP) concentration triggered by attachment of the cells to CU. We have now used a cellulose-coated polystyrene inducing the same cell behaviour to analyse the gap junction communication between aggregated cells. The transfer of the dye Lucifer Yellow (LY) between cells showed that cells aggregated on cellulose substratum rapidly (within 90 min) establish functional gap junctions. Inhibitors of cAMP protein kinase (PKI) or protein kinase C (GF109203X) both inhibited the diffusion of LY between neighbouring cells. Western blot analysis showed that this change in permeability was correlated with a decrease in Cx 43 phosphorylation. Thus, cellulose substrata seem to induce cell–cell communication through Cx 43 phosphorylation modulated by PKA and PKC. To understand the mechanisms by which a substratum regulates gap junctional communication is critically important for the emerging fields of tissue engineering and biohybrid devices.

Haemodialysis-induced pulmonary granulocyte sequestration in rabbits is organ specific.

Haemodialysis (HD) with cuprophan (CU) dialysers leads to a severe transient granulocytopenia. In the present study, we challenge the hypothesis that granulocytes sequester within the pulmonary vasculature simply because this is the first vascular bed encountered. This hypothesis is based upon experiments in which activated plasma or complement fragments were infused into animals, and may not pertain to the more complex HD situation. We used a rabbit model of HD, and returned the blood into the caval vein (v-HD) or aorta (a-HD). The mesentery was continuously monitored by intravital video microscopy, whereas other tissues were collected at the nadir of granulocytopenia and analysed immunohistochemically. Compared with controls, the number of granulocytes within alveolar walls was almost 2-fold higher following HD, with no difference between venous and arterial blood return. In addition, both v-HD and a-HD induced granulocyte accumulation within part of the larger pulmonary microvessels, though the amount of granulocytes found was 2-fold higher after v-HD. At no time did a-HD induce granulocyte sequestration within the mesenteric microcirculation. Neither did arterial return increase their number in other first-pass tissues like skeletal muscle or renal glomeruli, but it did so in the liver. In the heart, granulocyte content decreased during HD. Pulmonary sequestration of granulocytes during CU HD is not simply a first-pass effect, but is organ specific to a great extent. The accumulation within larger microvessels suggests an important role for adhesion molecules, whereas cellular stiffening may be involved in granulocyte retention within alveolar capillaries.

Effect of Membrane Composition on Cytokine Production and Clinical Symptoms During Hemodialysis: A Crossover Study

Background. Intradialytic symptoms including hypotension have been reported during dialysis and it has been suggested that these are related to the release of cytokines like IL-1β and TNFα by blood mononuclear cells when they get activated either due to contact with the dialyzer membrane or by compliment activation. Objective. To study the relationship between hemodialysis symptoms, cytokine production, and dialyzer membrane composition. Method. In a randomized prospective crossover study, 20 ESRD patients on maintenance hemodialysis were studied over cuprophan (CU) and polysulfone (PS) low flux dialyzer membranes for three weeks each undergoing a biweekly dialysis schedule of 4 h sessions. Serial IL-1β and TNFα levels were measured over 0, 15, 240 min during the first use of the dialyzer for all patients on both membranes. Intradialytic symptoms were monitored in a total of 240 dialysis sessions. Results. IL-1β levels increased from 16.6 ± 2.2 to 64.8 ± 25.1 pg/mL on CU and 21.5 ± 3.7 to 103.5 ± 30.7 pg/mL on PS membrane over the 4-h dialysis session. Similarly TNFα increased from 42.8 ± 4.5 to 354.9 ± 80.4 pg/mL on CU and 117.1 ± 53.7 to 387.0 ± 78.0 pg/mL on PS membrane. IL-1β levels increased significantly with PS membrane while TNFα rise was significant with both the membranes. Nausea was the most common symptom occurring in 138 dialysis sessions (57.5%). Vomiting, chest pain, fever, chills, and breathlessness occurred significantly more during dialysis with CU membrane as compared with PS membrane (P<0.01). Nausea, cramps, back pain, itching, restlessness, post dialysis fatigue, and hypotension did not differ between the two membranes. The mean rise in the cytokine levels during the first 15 min of sessions where the symptoms occurred, when compared with the mean rise in sessions where the symptoms did not occur, did not reveal any significant difference. Cytokine release did not correlate with the occurrence of intradialytic symptoms. Conclusion. Both CU and PS membranes increase circulating cytokine levels. More intradialytic clinical symptoms are seen in dialysis with CU as compared with PS membrane but the rise in cytokines IL-1β and TNFα does not appear to be responsible for them.

Dialysis-Membrane-Dependent Reduction and Adsorption of Circulating Hepatitis C Virus during Hemodialysis

Background/Aims: Patients on chronic hemodialysis (HD) are often infected with hepatitis C virus (HCV), a common cause of chronic liver disease. In some cases, however, decreases in the serum HCV load after HD have been documented. To better understand this phenomenon, we investigated the effects of various types of dialysis membrane on virus load in the circulation in vivo and in vitro. Methods: HCV RNA levels in patients’ serum, filtrate and dialyzer membranes were analyzed semiquantitatively by reverse transcription-polymerase chain reaction (RT-PCR) before and after HD treatment with two to four different types of dialysis membrane. HCV RNA was also determined from each fraction in in vitro dialysis and ultrafiltration. Results: In HD patients treated with a polysulfone (PS) membrane and a hemophan membrane, the HCV RNA titer reproducibly decreased by a factor of 10^–1–10^–2. In contrast, a cuprophan (CU) membrane had no detectable effect on HCV viremia, and HD with the AN69 membrane reduced HCV RNA levels in only a subset of the patients. In addition, a PS membrane, but not a CU membrane, reduced the level of circulating HCV in an in vitro assay. In both in vivo and in vitro experiments, HCV RNA was recovered from the PS membrane itself, but not from the ultrafiltrate. Conclusions: Membrane-dependent adsorption of HCV occurs during HD, causing a transient reduction in HCV in the circulation of patients.

Is Platelet-Activating Factor Produced during Hemodialysis with AN-69 Polyacrylonitrile Membrane?

Background: Platelet-activating factor (PAF) production during hemodialysis (HD) with cuprophane (CU) membrane has previously been demonstrated, while the results regarding PAF production during HD with AN-69 polyacrylonitrile membrane are dubious. In this study an attempt is made to show that PAF is produced during HD using AN-69 membrane while comparing this production with the corresponding one from HD with CU. Since previous studies have indicated that PAF, like the complement system, could also be implicated in HD-related leukopenia and thrombocytopenia (especially when CU membrane is used), the circulating leukocyte and platelet counts as well as the C3a-desArg and SC5b-9 (soluble, nonlytic form of the terminal complement complex) levels were measured. Methods: Ten hemodialyzed patients were subjected to HD with CU and AN-69 membranes for 2 consecutive weeks (first week with CU and second with AN-69). During the third HD session of each week and at different times (0, 2, 5, 15, 30, 60, 180 and 240 min), the PAF levels in the blood as well as the leukocyte and platelet counts were measured, while the circulating levels of the C3a-desArg and SC5b-9 were measured at 0, 5, 15, 60 and 240 min. PAF was detected by ethanol extraction, followed by purification by column chromatography and high-pressure liquid chromatography and finally quantified by bioassay. The C3a-desArg and SC5b-9 fractions of the complement were measured by immunoassay while an autoanalyzer gave the leukocyte and platelet counts. Results: Circulating PAF levels were detected at all time intervals during HD with AN-69 (PAF_AN-69) and CU (PAF_CU) membranes. At all time intervals PAF_AN-69 < PAF_CU, however, statistically significant differences (s) between the two membranes existed only at 15, 30, 60, 180 and 240 min. The highest PAF_AN-69 and PAF_CU occurred at 5 and 15 min into dialysis, respectively. The same observations were made for circulating C3a-desArg levels (s existed additionally at 5 min as well). The reduction of the circulating leukocytes had almost a mirror image with the C3a-desArg as well as PAF levels while the maximal reduction of platelets was observed after 2 min into dialysis with both membranes (i.e., simultaneously with the first increase in PAF secretion). Conclusions: PAF is indeed produced during HD with AN-69 membrane, as it is during HD with CU. At all time intervals during the HD procedure, PAF_AN-69 < PAF_CU. PAF seems to contribute to HD-related leukopenia and thrombocytopenia with both membranes.

The effect of LPS, uraemia, and haemodialysis membrane exposure on CD14 expression in mononuclear cells and its relation to apoptosis.

Both uraemia and bioincompatible haemodialysis membranes induce mononuclear cell apoptosis. Recent reports demonstrate that spontaneous apoptosis in normal monocytes is associated with the down-regulation of CD14 molecules, whereas LPS which prevents the down-regulation of CD14 favours monocyte survival. The aim of the present study was to evaluate a possible association between mononuclear cell apoptosis and low expression of CD14 molecules. This study also investigated whether LPS affects mononuclear cell CD14 expression and the apoptosis induced by uraemia and exposure to Cuprophan (CU) membrane. The study was performed in vitro examining the effects of CU membrane and LPS on mononuclear cells from normal subjects and from end-stage renal failure patients. Cells were analysed by flow cytometry with fluorescent monoclonal antibodies to determine CD14 expression and with Annexin-V labelling to determine apoptosis. In mononuclear cells from uraemic patients cultured for 48 h, there was a subset of cells with low CD14 expression; this subset of cells was not observed in normal monocytes cultured for the same period of time. Cells with low CD14 expression were also observed when normal or uraemic mononuclear cells were cultured in the presence of CU membrane. Simultaneous measurement of apoptosis and CD14 expression revealed that cells with low CD14 expression underwent apoptosis. The addition of LPS to the medium markedly reduced the number of mononuclear cells with low CD14 expression and also reduced the rate of apoptosis in these cells. Our data suggest that mononuclear cell apoptosis induced by uraemia and the CU membrane is associated with low CD14 expression. Furthermore, LPS prevented the decrease in CD14 and reduced the rate of apoptosis.

Effect of different dialyzers on defensins during hemodialysis

Background. Defensins are antimicrobial peptides that constitute 30% to 50% of total azurophil granule proteins. Although the activation of azurophils during hemodialysis has been reported, including the release of granulocyte lactoferrin, myeloperoxidase, and elastase, the release of defensins during hemodialysis has not been investigated. The purpose of this study was to determine the effects of hemodialysis on defensin release. Methods. Plasma defensin levels and excreted defensins in dialyzer eluates and dialysates during hemodialysis were determined by radioimmunoassay. Thirty hemodialysis patients were divided into three groups, each group using a different dialyzer membrane: cuprophan (CU), polymethylmethacrylate (PMMA), or polysulfone (PS). Results. The postdialysis plasma defensin level in the CU group was significantly increased compared with the predialysis level. In contrast, defensin levels in the PMMA and PS groups showed significant decreases postdialysis. The predialysis neutrophil count and defensin level exhibited a significant positive correlation in all groups. However, postdialysis, a significant positive correlation was found only in the CU group. Interestingly, defensin excretion in the dialyzer eluates of the PMMA group and in the dialysates of the PS group was higher than that in each of the other two groups respectively. Conclusions. No significant statistical correlation between postdialysis neutrophil count and defensin level was observed in the PMMA and PS groups, a finding that may be explained by the increased excretion of defensins during hemodialysis in these groups. These results suggested that plasma defensin level may be a marker for granulocyte activation in patients dialyzed with membranes showing lower levels of excreted defensins. This study also revealed the importance of determining both plasma changes and excreted volumes of granule proteins to define neutrophil degranulation during hemodialysis.

Studies of the Surface of Capillaries of Dialyzers with Atomic Force Microscopy

Microstructural properties of polymeric materials applied as membranes seem to be a potential factor influencing the biocompatibility of capillary dialyzers. The aim of our preliminary studies was the microscopic evaluation of the inner surface of cuprophane (CU) and polysulfone (PSU) membranes. Gambro GFE09 (CU) and Fresenius F4 (PSU) dialyzers were investigated using AFM ( Fig.1 and Fig.2 respectively ) and the optical measurement system (OMV) . The analysis of AFM images allowed to determine the average diameter of channels on the surface of the inner part of the dialyzer capillary (D) and the roughness (RMS) of the inner surface, which were as follows (in nm): Table 1. Table 1: D RMS Gambro GFE09 180 40 - 65 Fresenius F4 390 39 - 48 It can be concluded that the higher roughness and smaller, diameters of the channels in cuprophane may be the reason of the decreased biocompatibility of this membrane as compared to polysulfone.

Complement-dependent acceleration of apoptosis in neutrophils by dialyzer membranes

Recently, we have shown that cuprophan (CU) causes receptor modulation by a C5-dependent mechanism, which is activated by neutrophil-derived reactive oxygen intermediates. The objective of our study was to evaluate the contribution of dialyzer membranes to the induction of apoptosis in human neutrophils [polymorphonuclear cells (PMNs)]. PMNs harvested from healthy donors were incubated with hollow fibers from a biocompatible membrane polysulfone (PS) and a bioincompatible membrane CU, all in the presence of 25% human serum. After 4, 8, and 12 hours of incubation at 37 degrees C, apoptosis was quantitated by counting the numbers of cells showing features of apoptosis on cytospins by light microscopy and also by flow cytometry using propidium iodide nuclear staining. Compared with PMNs incubated with serum alone, cells cultured with fibers of PS demonstrated a higher percentage of apoptosis. Fibers from CU dialyzers led to a more pronounced induction of apoptosis in PMNs, which was significantly higher compared with PS. This effect was partly mediated by heat-sensitive serum products and depended on the presence of divalent cations. In contrast to the recently described C5-dependent pathway in PMN receptor modulation by CU, this effect seemed to depend on the presence of the complement factor C3. In conclusion, our results indicate that besides the well-known accelerated apoptosis of PMNs in uremia, both biocompatible and bioincompatible dialyzer material itself can accelerate apoptosis in human PMNs.

Diffusion and calibration properties of microdialysis sampling membranes in biological media.

The diffusion and calibration properties for three commercially available microdialysis membranes (polycarbonate-polyether (PC), polyacrylonitrile (PAN), and cuprophan (CUP)) were evaluated. The analytes studied had molecular weights between 94 (phenol) and 1355 (vitamin B12). For each analyte-membrane pair, an effective membrane diffusion coefficient was calculated. Effective membrane diffusion coefficients varied considerably between the microdialysis membranes. For Vitamin B12, CUP and PAN membranes gave relative recovery values of greater than 20% at 0.5 microl min(-1), while the PC membrane had a 1% recovery. When backpressure was applied. PC and PAN membranes exhibited more ultrafiltration than CUP membranes. Ultrafiltration did not affect analyte relative recovery through either PC or PAN membranes. Effective membrane diffusion coefficients were not significantly altered for some membrane-analyte combinations when exposed to 4% bovine serum albumin or 0.3% fibrinogen. These data suggest that reductions in relative recovery during long-term microdialysis sampling experiments may be due to other physiologically relevant proteins or to tissue reactions near the dialysis membrane.

IN VIVO INTRACELLULAR CYTOKINE PRODUCTION BY LEUKOCYTES DURING HAEMODIALYSIS

Several chronic inflammatory changes undergone during chronic haemodialysis are associated with increased pro-inflammatory cytokine production. Although generation of anaphylatoxins has been incriminated in the untoward effects of haemodialysis, it is still debated whether anaphylatoxins stimulate monocyte secretion of TNFα and IL-1. We demonstrate that peripheral mononuclear cells isolated from healthy controls and cultured with complement-activated autologous serum or recombinant C5a induced high levels of IL-1, IL-1ra, IL-8 and MCP-1, low levels of TNFα and sTNFRII but no IL-10 and MIP-1α. Cytokine production by leukocytes was investigated by FACS analysis in six patients dialysed consecutively with three equivalent low permeability membranes known to activate the complement to different degrees: polysulfone (F6HPS), cellulose acetate (CA) and cuprophane (CP). Percentage of leukocytes expressing IL-1, IL-1ra, TNF-α and IL-8 is increased in patients dialysed with CP. Moreover, we show for the first time that haemodialysis is associated with the production of cytokines by circulating neutrophils. Predialysis plasma levels of MCP-1 and TNFRII did not increase during the dialysis session at the time when anaphylatoxin generation was highest. Dialysis with membranes that activate the complement to a high extent induce activation of leukocytes which may explain chronic complications associated with dialysing with CP.

Activation of the cyclic AMP pathway in cells adhering to biomaterials: regulation by vitronectin– and fibronectin–integrin binding

Our previous studies have shown that cells adhering to biomaterials in serum-free conditions increase their content of cyclic AMP (cAMP) and become aggregated. In cells on an acrylonitrile membrane (AN69), these biochemical and morphological changes are prevented by adding 10% foetal calf serum (FCS) to the medium; cells on the cellulose membrane Cuprophan (CU) remain unaffected. The present study examines the roles of vitronectin (VN)– and/or fibronectin (FN)–integrin binding in this inhibition. Competitively blocking VN- and FN-receptors with echistatin increased intracellular cAMP significantly and caused cells on AN69 to aggregate, but did not modify cAMP-dependent cell aggregation on CU. VN or FN adsorbed onto CU also inhibited cAMP production by attached cells and prevented their aggregation, whereas adsorbed BSA had no effect. Therefore, the binding of VN or FN to cell-surface integrins seems to limit the activation of the cAMP pathway initiated by the substratum itself.

Biocompatibility of MPC: in vivo evaluation for clinical application

Biocompatibility is important to assure a mild body reaction to an implanted device and its long-term stability and functionality. In diabetes research, subcutaneously implanted glucose monitoring systems need biocompatible surfaces for long-term application. The biocompatibility of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (MPC), a material similar to the phospholipid layer of a cell membrane, was compared in vivo with the biocompatibility of polyurethane (PU), polyvinyl alcohol (PVA), and cuprophane (CUP). Needle-type glucose sensors and hollow-fiber probes used for microdialysis were coated with these four different biomaterials and implanted subcutaneously in 18 rats and 7 healthy volunteers. At set intervals, the implants and, in the case of the rats, also the surrounding tissue were removed and characterized by light and electron microscopy. MPC-coated sensors and hollow-fiber probes showed smooth and thin deposits in flat layers, whereas the surface deposits on PU- and PVA-coated sensors and those on CUP hollow-fiber probes appeared as rough, irregular, and dense attachments of aggregated cells and protein. This study confirmed results from earlier in vitro tests by showing the biocompatibility and reliability of MPC. Even though the amount of protein and cells attached to the MPC surface was not as low as expected from in vitro experiments, the biocompatibility and long-term stability of the implanted devices were superior to those of PU, PVA, and CUP.

Ex vivo Flow Cytometry Determination of Intracytoplasmic Expression of IL-2, IL-6, IFN-γ, and TNF-α in Monocytes and T Lymphocytes, in Chronic Hemodialysis Patients

Aims: To determine the intracytoplasmic expression of TNF-α, IL-2, IL-6 and IFN-γ, ex vivo and in vitro, in both monocytes and T lymphocytes by flow cytometry after appropriate stimulation using phorbol myristate acetate (PMA)/ionomycin or lipopolysaccharides (LPS) in the presence of monensin, in order to assess the bio(in)compatibility of different dialysis membranes. Methods: We examined monocytes and T lymphocytes taken from chronic hemodialysis patients (using either cuprophane (CUP), n = 6; polyacrylonitrile (AN 69), n = 6; or polysulfone (PS), n = 6 membranes), before and after a dialysis session. We compared the results with those obtained from end-stage chronic renal failure patients (n = 3) and healthy volunteers (n = 11). Results: Before any stimulation there was a statistically significant difference in the percentages of TNF-α, IL-6, and IFN-γ- expressing monocytes with respect to the dialysis membrane used. The highest percentages were observed for CUP and AN69 patients with figures of around 30% for each cytokine; the lowest percentages were found in PS patients and healthy volunteers. One hour after LPS stimulation the patterns remained unchanged for TNF-α and IFN-γ, whereas the percentages of IL-6-expressing cells in PS patients and in healthy volunteers reached the figures obtained in the other groups. When we examined the percentage of IFN-γ-, TNF-α- and IL-6-expressing monocytes in patients before and after a dialysis session, before any stimulation, we found that the results were significantly different for the three membranes (p = 0.01). Thus, a dialysis session with polysulfone membranes had no significant effect on the precentages of IFN-γ-, TNF-α-, and IL-6-expressing monocytes, whereas percentages were significantly lower after the dialysis session when using cuprophane or AN69 membranes, suggesting a release of these cytokines by the monocytes during dialysis. A significant number of IFN-γ- and IL-2-expressing T lymphocytes were only detected after 18 hours of PMA/ionomycin stimulation. The percentages of IFN-γ-expressing T cells recorded for the different membranes were not statistically different from those recorded for healthy subjects or pre-dialysis patients, i.e., they were between 11.5 and 20%. However, the percentages of IL-2-expressing T lymphocytes were significantly different between the 5 groups, i.e., 31.3, 30.5, 18.6, 13.9 and 7.6%, respectively, for CUP patients, pre-dialysis patients, healthy volunteers, PS and AN69 patients. This suggests that pre-dialysis and CUP patients have, at baseline, a stimulation of their T lymphocytes. Finally, a 4-hour dialysis session had no impact on the percentages of IL-2-expressing T lymphocytes, whereas it was associated with a significant decrease in the percentage of IFN-γ-expressing cells, but only when cuprophane membranes were used. Conclusion: Cytokine flow cytometry enables one to study, ex vivo, i.e., without any stimulation of the cells, and in vitro after appropriate stimulation, the bio(in)compatibility of dialysis membranes assessed by the intracytoplasmic cytokine profiles of TNF-α, IFN-γ, IL-6 and IL-2, evaluated at the single cell level.