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PubMed1997

Acquired C3 deficiency in patients with alcoholic cirrhosis predisposes to infection and increased mortality.

BACKGROUND: Acquired deficiencies of certain complement proteins and impaired opsonisation activity have been implicated in the pathogenesis of the increased susceptibility to infections of patients with alcoholic cirrhosis. METHODS: Serum concentrations of C3 and C4, plasma concentrations of C3bc, C9, and the terminal C5b-9 complement complex (TCC), and haemolytic complement activity (classic and alternative pathway) of serum, and serum opsonic activity were determined in 46 patients with compensated alcoholic cirrhosis, 31 who were decompensated, and in 15 healthy subjects. After 19 months (median) the investigated variables were analysed for their use in prognosis of recurrent infections and survival. RESULTS: C3 and C4 concentrations and the haemolytic complement activity of the alternative pathway were decreased in decompensated cirrhotic patients compared with controls (p < 0.01). Univariate analysis (log rank test) showed that low concentrations (< or = lower quartile) of C3 (p < 0.001) and C3bc (p < 0.05), haemolytic complement activity of the alternative pathway (p < 0.01) and classic pathway (p < 0.05), and decompensated cirrhosis (p < 0.001) were associated with an increased risk of infection and increased mortality. Multivariate (Cox) analysis showed that low C3 concentrations and decompensation of cirrhosis were significant predictors of infections and mortality (p < 0.02). CONCLUSIONS: Low serum C3 concentrations and decreased haemolytic complement function predisposes to infection and increased mortality in patients with alcoholic cirrhosis.

Adult↗
PubMed1986

Repolarization of the membrane potential of blood platelets after complement damage: evidence for a Ca++ -dependent exocytotic elimination of C5b-9 pores.

Gel-filtered blood platelets exposed to complement proteins C5b-9 have previously been shown to undergo a reversible depolarization of membrane potential (Em) in the absence of lytic plasma membrane rupture. In this paper, we examine the mechanism by which C5b-9 damaged platelets restore their basal electrochemical state, despite increased ion conductance due to membrane insertion of these cytolytic serum proteins. Repolarization of Em after formation of the C5b-9 membrane pore is shown to be accompanied by a Ca++-dependent vesiculation of the platelet surface, which results in the release of these proteins from the plasma membrane and a restoration of the membrane's functional integrity. This exocytotic elimination of C5b-9 complexes from the plasma membrane is accompanied by a ouabain-inhibitable repolarization of Em, which presumably reflects restoration of transmembrane cation gradients by the plasma membrane Na/K ATPase. The role of external Ca++ in the platelet's response to membrane-insertion of the C5b-9 proteins is discussed both in the context of the known cellular effects of this ion and in the context of recent observations suggesting sublytic changes in platelet function after complement-mediated plasma membrane damage.

Adult↗
PubMedDate not supplied

Induction of matrix protein synthesis in human glomerular mesangial cells by the terminal complement complex.

Exposure of human glomerular mesangial cells (GMC) in culture to sublytic doses of the terminal complement proteins C5b-8 and C5b-9 caused a transient increase in abundance of mRNA specific for collagen type IV and fibronectin; mRNA of laminin was not affected. Since C5b-9 is found deposited in inflamed or sclerotic areas we propose that stimulation of matrix protein synthesis by C5b-9 might contribute to the development of glomerular sclerosis.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1984

Species-restricted target cell lysis by human complement: complement-lysed erythrocytes from heterologous and homologous species differ in their ratio of bound to inserted C9.

The cytolytic efficiency of the terminal complement complex (C5b-9) against erythrocytes of different species is, in part, dependent on the species of C9 origin. In the present study, we have examined the interaction of C9 with erythrocytes in terms of the binding, dimerization, and insertion of C9 into the membranes of sheep and human erythrocytes lysed by human complement (C). The membranes of these C-lysed erythrocytes were analyzed for bound, dimerized, and inserted C9 by a combination of photolabeling, SDS-PAGE, electroblotting, and immunostaining techniques. We found that neither binding nor dimerization of C9 could be correlated with the relative hemolytic efficiency of human C on these erythrocytes, but that C9 insertion into the membranes of these cells varied in direct relation to the extent of lysis. Interestingly, the binding of C3 to these cells under conditions of equivalent C1 fixation also correlated with lytic efficiency. These data indicate that the C9-related differences in the cytolytic efficiency of C against erythrocytes from different species is primarily due to the efficiency of C9 insertion into these cells. Moreover, these data emphasize that neither the binding of C9 to a target membrane nor the formation of C9 dimers necessarily leads to the insertion of C9 into the membrane, suggesting the presence of membrane-bound but inactive C5b-9 complexes. Because the extent of C3 binding also correlated with the relative degree of lysis of sheep vs human erythrocytes, the possibility exists that surface-bound C3 may regulate hemolysis by directing the insertion of C9 in terminal complexes into cells.

Animals↗
PubMed1981

Direct measurement of the increase in intracellular free calcium ion concentration in response to the action of complement.

1. The effect of rabbit anti-(pigeon erythrocyte) antibodies plus human complement on the concentration of intracellular free Ca2+ in sealed pigeon erythrocyte 'ghosts' was investigated with the photoprotein obelin. 2. The addition of human serum, as a source of complement, to 'ghosts' coated with antibody caused a rapid increase in intracellular free Ca2+ after a lag of 20-40 s, as detected by an increase in obelin luminescence. 3. The increase in obelin luminescence could not be explained by release of obelin into the medium. It was also Ca2+-dependent in that extracellular EGTA abolished the effect and intracellular EGTA inhibited it and required the complete terminal complex (C56789). No effect was seen with C5678. 4. The concentration of intracellular free Ca2+ before addition of complement was approx. 0.3 microM. This increased to a maximum of 5-30 microM after complement addition and then remained constant for at least 1-2 min. 5. Antibody plus complement induced a rapid increase in 42K+ efflux and an inhibition of cyclic AMP formation. 6. When partially purified complement components (C5b-9) were used in 'reactive lysis' it was possible to inhibit the release of macromolecules from pigeon erythrocyte 'ghosts' by extracellular EGTA. 7. It was concluded that the increase in intracellular free Ca2+ concentration caused by anti-cell antibody plus complement occurred before cell lysis and may be involved in the mechanism of complement-induced cell injury.

Animals↗
PubMed1981

Membrane attack complex of complement: distribution of subunits between the hydrocarbon phase of target membranes and water.

Membrane destruction by complement is effected by the membrane attack complex (MAC) which is the dimer of a fusion product of the complement proteins C5b, C6, C7, C8, and C9. Phospholipid bilayer vesicles were used as target membranes for the MAC and its intermediate complexes. The subunits of these membrane-bound complexes were explored as to their relative exposure to the hydrocarbon phase of the lipid bilayer and to water surrounding the lipid vesicles. Protein exposed to the aqueous phase was labeled with 125I; protein exposed to the hydrocarbon phase was labeled by using tritiated azido phospholipids and irradiation. Analysis of the membrane-bound MAC showed that subunits C5b, C8 beta, and C9 were exposed to the aqueous phase. The subunits C8 alpha-gamma and C9 were primarily in contact with the hydrocarbon phase. C6 and C7 were little exposed to either phase, suggesting that these proteins are inaccessible within the MAC. Analysis of the intermediate complexes showed that C5b was the subunit most exposed to water in membrane-bound C5b-7, and C5b and C8 beta were the water-exposed subunits in C5b-8. Subunit exposure to the hydrocarbon phase of the lipid bilayer changed during MAC assembly. Whereas all three subunits of C5b-7 carried the phospholipid photolabel; most of the label was bound to the C8 subunit in C5b-8 and to C9 in the MAC. It is proposed that contact with the hydrocarbon core of membranes is established by C5b-7 through each of its subunits, by C5b-8 through C8, and by the MAC through C8 and, particularly, C9.

Affinity Labels↗
PubMed1987

Bacterial killing by complement. C9-mediated killing in the absence of C5b-8.

The ability of serum complement to kill Gram-negative bacteria requires assembly of the membrane attack complex (MAC) on the cell surface. The molecular events that lead to cell killing after MAC assembly are unknown. We have investigated the effect of C9 on bacterial survival in the presence and absence of its receptor, the C5b-8 complex, on the outer membrane. A fluorescence assay of the membrane potential across the inner bacterial membrane revealed that addition of C9 to cells bearing the performed C5b-8 complex caused a rapid and complete dissipation of the membrane potential. No fluorescence change was observed in serum-resistant strains of Escherichia coli. Addition of trypsin, after C9 was bound to C5b-8, did not rescue the cells from the lethal effects of C9. Furthermore, assays of cell killing kinetics and C9 binding indicate that formation of tubular poly(C9) is not required for killing. When C9 was introduced into the periplasmic space in the absence of its receptor by means of an osmotic shock procedure, cell killing occurred. Other proteins, such as C8 or serum albumin, were not toxic, and C9 was ineffective against two resistant strains. The results presented here and previously [Dankert & Esser (1986) Biochemistry 25, 1094-1100], when considered together, indicate that the 'lethal unit' in complement killing of some Gram-negative bacteria is a C9-derived product that acts by dissipation of cellular energy.

Blood Bactericidal Activity↗
PubMed1979

C5b-9 dimer: isolation from complement lysed cells and ultrastructural identification with complement-dependent membrane lesions.

The membrane attack complex (MAC) of complement was extracted from the membranes of cells lysed by human complement and its properties were compared with those of the fluid phase complex SC5b-9. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunochemical analysis, the two isolated complexes had identical subunit compositions, except that the MAC lacked the S-protein. The sedimentation coefficient and molecular weight of the extracted and isolated MAC were, respectively, 33.5 S and 1.7 x 10(6) daltons, compared to 23 S and 1.0 x 10(6) dalton for SC5b-9. Because the molecular weight of the MAC is approximately two times greater than that of C5b-0 (800,000 daltons), the MAC is considered the dimer of C5b-9. Under specified conditions, the 33.5 S dimer could be converted to the 23 S monomer without dissociation of subunits. The MAC had the electron microscopic appearance and dimensions that are characteristic for the complement produced ultrastructural membrane lesions. SC5b-9 had a different ultrastructure that is dissimilar to the morphology of the lesions. The isolated MAC could be reincorporated into phospholipid bilayers and assumed on the surface of the resultant lipid vesicles the orientation and appearance of typical complement lesions.

Animals↗
PubMed1999

Endothelial nuclear factor-kappaB translocation and vascular cell adhesion molecule-1 induction by complement: inhibition with anti-human C5 therapy or cGMP analogues.

We have previously shown that reoxygenation of hypoxic human umbilical vein endothelial cells (HUVECs) leads to the activation and deposition of complement. In the present study, we investigated whether the terminal complement complex (C5b-9) influences HUVEC nuclear factor-kappaB (NF-kappaB) translocation and vascular cell adhesion molecule-1 (VCAM-1) protein expression after hypoxia/reoxygenation by decreasing endothelial cGMP. Additionally, we investigated the action of anti-human C5 therapy on endothelial cGMP, NF-kappaB translocation, and VCAM-1 protein expression. Reoxygenation (0.5 to 3 hours, 21% O(2)) of hypoxic (12 hours, 1% O(2)) HUVECs in human serum (HS) significantly increased C5b-9 deposition, VCAM-1 expression, and NF-kappaB translocation compared with hypoxic/reoxygenated HUVECs treated with the recombinant human C5 inhibitor h5G1.1-scFv. Acetylcholine (ACh)-induced cGMP synthesis was significantly higher in normoxic HUVECs compared with hypoxic HUVECs reoxygenated in HS but did not differ from hypoxic HUVECs reoxygenated in buffer or HS treated with h5G1.1-scFv. Treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv or cGMP analogues significantly attenuated NF-kappaB translocation and VCAM-1 protein expression. Treatment with NO analogues, but not a cAMP analogue, cGMP antagonists, or an NO antagonist, also significantly attenuated VCAM-1 expression. We conclude that (1) C5b-9 deposition, NF-kappaB translocation, and VCAM-1 protein expression are increased in hypoxic HUVECs reoxygenated in HS; (2) reoxygenation of hypoxic HUVECs in HS, but not buffer alone, attenuates ACh-induced cGMP synthesis; and (3) treatment of hypoxic/reoxygenated HUVECs with h5G1.1-scFv attenuates C5b-9 deposition, NF-kappaB translocation, and VCAM-1 expression while preserving ACh-induced cGMP synthesis. C5b-9-induced VCAM-1 expression may thus involve an NO/cGMP-regulated NF-kappaB translocation mechanism.

Aminoquinolines↗
PubMed1990

Role of calcium and calpain in complement-induced vesiculation of the platelet plasma membrane and in the exposure of the platelet factor Va receptor.

The role of calcium and intracellular calpains in the expression of platelet prothrombinase activity was investigated. Incubation of gel-filtered platelets with complement proteins C5b-9 resulted in alpha-granule and dense granule secretion and exposure of membrane binding sites for coagulation factors Va and Xa. This was accompanied by the release of microparticles from the cell surface that incorporated plasma membrane glycoproteins GP Ib, IIb, and IIIa and the alpha-granule membrane protein GMP-140. Generation of these membrane microparticles was dependent on the presence of extracellular calcium and was accompanied by proteolytic degradation of the cytoskeletal proteins, actin binding protein (ABP), talin, and myosin heavy chain. Microparticle formation was also detected when unstirred platelets were activated by thrombin plus collagen, although proteolysis of ABP, talin, or myosin was not observed. Preincorporation of the calpain inhibitor leupeptin into the platelet cytosol completely blocked C5b-9-induced proteolysis of ABP, talin, and myosin. However, inhibition of this calpain-mediated proteolysis had no effect on platelet secretion, the generation of microparticles, the exposure of membrane sites for factors Va and Xa, or the expression of prothrombinase activity. Furthermore, the microparticles that formed in the presence of leupeptin contained intact ABP, talin, and myosin heavy chain. Prior depletion of ATP with metabolic inhibitors eliminated all platelet responses to thrombin plus collagen, but did not affect C5b-9-induced microparticle formation or exposure of binding sites for factor Va on the microparticles. These data indicate that the formation of microparticles and the expression of platelet prothrombinase activity in response to C5b-9 are dependent upon an influx of calcium into the platelet cytosol, but do not require metabolic energy or calpain-mediated proteolysis of cytoskeletal proteins.

Adenosine Triphosphate↗
PubMed1992

Humoral mechanisms in immune neuropathies.

Antibody and complement are implicated in the pathogenesis of a number of human, primarily demyelinating neuropathies. The ability of serum and purified, primarily IgM, antibodies to mediate demyelination was demonstrated in both in vitro and in vivo model systems. Complement activation to produce channel-forming terminal complement complexes, C5b-8 and C5b-9, was required for demyelination in vitro. Antibodies implicated in the demyelination of peripheral nerve of GBS patients and patients with monoclonal gammopathy-associated neuropathy bind carbohydrate epitopes on various neutral or acidic glycolipids and glycoproteins of peripheral nerve. In acute monophasic GBS, antibodies of multiple specificities may be induced to different infectious agents. These Ab, following penetration of a damaged blood-nerve barrier, are proposed to bind determinants of human peripheral nerve and participate in demyelination of nerve through activation of complement. These antibodies correlate with the clinical course, the generation of complement activation products, and the response to plasmapheresis. The mechanism by which the blood-nerve barrier is broken in GBS and other inflammatory demyelinating neuropathies and the extent of the role of the cellular immune system remain to be determined. Recent experiments demonstrated that T cells, antibody, and complement could synergistically contribute to central nervous system demyelination in naive rats. A similar synergism would be an attractive hypothesis for demyelination in the peripheral nervous system.

Autoantibodies↗
PubMed1986

Assembly of the membrane attack complex of complement on small unilamellar phospholipid vesicles.

Light-scattering intensity was shown to be a reliable, direct, and quantitative technique for monitoring the assembly of the membrane attack complex of complement (proteins C5b-6, C7, C8, and C9) on small unilamellar phosphatidylcholine vesicles. The assembly on vesicles occurred in a simple fashion; complexes of C5b-7 bound noncooperatively to the vesicles, and final assembly of C5b-9 did not induce vesicle aggregation or fragmentation. When C5b-6 and C7 were mixed in the presence of vesicles but at molar protein/vesicle ratios of less than 1, there was quantitative binding of C5b-7 to the vesicles with no concomitant aggregation of C5b-7. If C7 was added at a slower rate, quantitative binding was obtained at molar C5b-7/vesicle ratios of up to 5. The latter observations (a) were consistent with the proposal that C5b-7 aggregation and membrane binding were competitive events and (b) defined conditions under which light-scattering intensity measurements could monitor C5b-9 assembly on vesicles without contribution from the fluid-phase assembly. The C8/C5b-7 ratio in the phospholipid-C5b-8 complex was 0.97 +/- 0.12, and the maximum ratio of C9/C5b-8 in the final complex was 16.2 +/- 2.0. One C9 molecule associated rapidly with each phospholipid-C5b-8, followed by slower incorporation of the remaining C9 molecules. The initial velocity of the slow phase of C9 addition was easily saturated with C9 and gave an activation energy of 37 kcal/mol. This was identical with the value measured for the analogous process in the fluid-phase assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement C7↗
PubMed2001

Human serum-induced expression of E-selectin on porcine aortic endothelial cells in vitro is totally complement mediated.

BACKGROUND: Whereas complement is a key mediator of hyperacute xenograft rejection, its role in acute vascular rejection (AVR) is a matter of controversy. AVR is associated with de novo synthesis of endothelial cell-derived inflammatory mediators, including the leukocyte-recruiting adhesion molecule E-selectin. Here we investigate the role and mechanism of complement in human serum-induced porcine endothelial cell activation. METHODS: An in vitro xenotransplantation method was designed using porcine aortic endothelial cells stimulated with human serum in microculture wells. E-selectin expression was measured by cell-enzyme immunoassay. Complement inhibitors acting at different levels in the cascade were investigated for their effect on E-selectin expression. RESULTS: E-selectin was strongly induced by normal human serum but not by heat-inactivated serum. Compstatin, a synthetic C3 inhibitor, markedly reduced human serum-induced E-selectin expression. Purified C1-inhibitor suppressed E-selectin induction completely, indicating activation through the classical or lectin pathway. Furthermore, a monoclonal antibody (mAb) that inhibits cleavage of C5 or another mAb that blocks the function of C7, completely inhibited the expression of serum-induced E-selectin, consistent with the terminal C5b-9 complement complex being the mediator of the endothelial cell activation. Inhibition of the alternative pathway using a novel antifactor D mAb did not reduce E-selectin expression. CONCLUSION: Human serum-induced expression of porcine E-selectin is totally complement dependent, induced by a C1-inhibitor regulated pathway and mediated through the terminal complement complex. The data may have implications for therapeutic strategies, particularly of C1-inhibitor and anti-C5 mAb, to protect against endothelial cell activation and subsequent AVR of porcine xenografts.

Animals↗
PubMed1991

Ultrastructural and biochemical observations on antineutrophil antibody- and complement-induced immuno-injury to equine neutrophils.

Antibody-induced damage to neutrophils was studied to elucidate processes associated with destruction of neutrophils in immune-mediated neutropenias. Cytomorphological changes and release of certain cellular constituents were determined for neutrophils treated with an antineutrophil serum in the presence or absence of rabbit complement. Neutrophils exposed to the antineutrophil serum alone showed endocytotic vacuoles and degranulation. In contrast, neutrophils exposed to the antineutrophil serum and complement showed marked morphologic changes. The plasma membrane developed numerous vesicles, villous processes and minute areas of bilayer discontinuity. Highly damaged cells exhibited cellular and nuclear swellings, disruption of cytoplasmic integrity and disordered distribution of lysosomal granules. Cytoplasmic constituents (K+ and lactate dehydrogenase) were released extracellularly from neutrophils exposed to the antineutrophil serum with or without complement. Cytological changes induced by the antineutrophil serum and complement were analogous to those reported for leucocytes exposed to the activated complement components C5b-9 (the membrane attack complex) and bacterial toxins. It was concluded that the cytological abnormalities observed were most probably associated with immune-mediated damage to the cell membrane, leading to leakage of cytoplasmic constituents like K+, colloidal osmotic swelling, and disruption of the cytoskeletal system.

Alkaline Phosphatase↗
PubMed1999

C6 produced by macrophages contributes to cardiac allograft rejection.

The terminal components of complement C5b-C9 can cause significant injury to cardiac allografts. Using C6-deficient rats, we have found that the rejection of major histocompatibility (MHC) class I-incompatible PVG.R8 (RT1.A(a)B(u)) cardiac allografts by PVG.1U (RT1.A(u)B(u)) recipients is particularly dependent on C6. This model was selected to determine whether tissue injury results from C6 produced by macrophages, which are a conspicuous component of infiltrates in rejecting transplants. We demonstrated that high levels of C6 mRNA are expressed in isolated populations of macrophages. The relevance of macrophage-produced C6 to cardiac allograft injury was investigated by transplanting hearts from PVG. R8 (C6-) donors to PVG.1U (C6-) rats which had been reconstituted with bone marrow from PVG.1U (C6+) rats as the sole source of C6. Hearts grafted to hosts after C6 reconstitution by bone marrow transplantation underwent rejection characterized by deposition of IgG and complement on the vascular endothelium together with extensive intravascular aggregates of P-selectin-positive platelets. At the time of acute rejection, the cardiac allografts contained extensive perivascular and interstitial macrophage infiltrates. RT-PCR and in situ hybridization demonstrated high levels of C6 mRNA in the macrophage-laden transplants. C6 protein levels were also increased in the circulation during rejection. To determine the relative contribution to cardiac allograft rejection of the low levels of circulating C6 produced systemically by macrophages, C6 containing serum was passively transferred to PVG.1U (C6-) recipients of PVG.R8 (C6-) hearts. This reconstituted the C6 levels to about 3 to 6% of normal values, but failed to induce allograft rejection. In control PVG.1U (C6-) recipients that were reconstituted with bone marrow from PVG.1U (C6-) donors, C6 levels remained undetectable and PVG.R8 cardiac allografts were not rejected. These results indicate that C6 produced by macrophages can cause significant tissue damage.

Animals↗
PubMed1997

Effects of cardiopulmonary bypass on neonatal and paediatric inflammatory profiles.

OBJECTIVE: Cardiopulmonary bypass (CPB) causes significant morbidity in paediatric patients, yet the mechanisms involved in the related inflammatory processes (resulting in capillary leak and edema) are poorly understood. Moreover, earlier palliative and corrective intervention in neonates and infants has provided the cohorts of patients about whom little is known of their proinflammatory response. METHODS: In the present two group study, 14 neonates (age 1-28 days, 2.5-4.5 kg) and 13 infants (2-12 months, 3-7 kg), undergoing CPB for congenital heart disease were consecutively recruited. The two cohorts were well matched in terms of CPB and aortic cross-clamp times (P > 0.1). Blood samples were collected on induction of anaesthesia, 5 min following onset of CPB, at the end of CPB, and 30 min, 2 and 24 h post-protamine (PP) administration. Plasma concentration of cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8), terminal complement complex (C5b-9) neutrophil counts and leucocyte elastase were measured. RESULTS: Plasma levels of all inflammatory markers significantly increased in both groups during and following CPB as compared to baseline. During and following CPB the change in IL-8 level was more pronounced in neonates (peak 30 min PP, median(range): 1062 (182-3872) pg/ml) than in infants 568 (172-1368) pg/ml), P = 0.01. Changes in IL-6 level were indistinguishable between groups intraoperatively, but remained significantly higher at 24 h in neonates (P = 0.02). Peri and postoperative levels of C5b-9 were significantly higher in infants than in neonates (peak 30 min PP, median (range): 984 (118-1142) ng/ml vs 458 (22 1340) ng/ml in neonates respectively, P = 0.01) but were similar at 24 h. Despite this, leucocyte elastase profiles did not differ significantly between the respective cohorts. CONCLUSION: These results indicate that there may be differences between neonates and infants with regard to the inflammatory response to CPB and neonatal patients merit further investigation in order to elucidate whether the pathophysiology of their CPB related inflammatory response and its clinical sequelae differs from their older counterparts.

Cardiopulmonary Bypass↗
PubMed2000

Biocompatibility of silicone-coated oxygenator in cardiopulmonary bypass.

BACKGROUND: This study was designed to analyze the biocompatibility of silicone-coated oxygenators using inflammatory response as the outcome measure, and to investigate whether the silicone-coated oxygenators perform better in terms of postoperative organ dysfunction. METHODS: The 32 patients who underwent cardiopulmonary bypass (CPB) were divided into 3 groups: group A (n = 10), heparin-coated circuit with silicone-coated oxygenator; group B (n = 11), whole heparin-coated circuit; and group C (n = 11), whole untreated circuit. The plasma concentrations of the proinflammatory markers, made of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1beta, interleukin-6, interleukin-8), terminal complement complex (C5b-9), and polymorphonuclear elastase (PMN-E), were measured by enzyme-linked immunosorbant assay. RESULTS: All proinflammatory markers were significantly lower in groups A and B than in group C, especially C5b-9 and PMN-E concentrations, which were significantly lower in group A than in group B. The alveolar-arterial oxygen gradients (A-aDO2) and the respiratory index were significantly better in group A than in group C. In group B, however, only the A-aDO2 was significantly better than in group C. The duration of intubation and the length of stay in the intensive care unit stay were significantly shorter in groups A and B than in group C. CONCLUSIONS: Silicone-coated oxygenators are biocompatible and prevent postoperative organ dysfunction.

Anticoagulants↗
PubMed1997

A flow cytometric immunoassay to quantify adsorption of complement activation products (iC3b, C3d, SC5b-9) on artificial surfaces.

Crosslinked agarose microspheres and various polystyrene microspheres were analyzed for complement components after incubation with serum at 37 degrees C for times up to 2 h. Quantification involved direct flow cytometric analysis of the beads after the bound complement proteins were indirectly fluorescently tagged by use of a monoclonal antibody against a complement protein: C5b-9, iC3b, C3d, C4d, Bb, C3a, and C1q. Calibration with fluorescein microbead standards demonstrated that the membrane attack complex (SC5b-9) was surface bound on all surfaces and that the surface concentration gradually increased to levels as high as 0.5 micrograms/cm2. Further, the surface bound represented a substantial percentage of the total generated. The iC3b level on polystyrene beads rapidly reached 0.09 micrograms/cm2 and the C3d levels were an order of magnitude less. On agarose beads the iC3b levels continually rose to 0.17 micrograms/cm2 and, as before, the C3d levels were substantially lower. The surface concentration of C4d and Bb on both surfaces were significant but less than 1.0 ng/cm2. There was minimal evidence of C3a and C1q adsorption for any surface. Use of amino-polystyrene beads moderately reduced the level of bound iC3b, C3d, and SC5b-9, whereas carboxylated beads reduced the levels by almost a factor of two. The appreciable amounts of iC3b and SC5b-9 consistently noted on the artificial surfaces tested in this paper suggests that for these two activation products in vitro analysis of material induced complement activation should also include surface analysis.

Adsorption↗