Ulf R. Nilsson

A wettability gradient method for studies of macromolecular interactions at the liquid/solid interface

A method to make a surface energy gradient along silicon or glass plates, 20 mm long, has been developed. The plates are hydrophobic at one end and hydrophilic at the other with a gradient of increasing wettability in between. The wettability gradient has a sigmoidal character and can be visualized by the use of a capillary rise method. With the use of ellipsometry with sufficient lateral resolution, it is then possible to study quantitative aspects of macromolecular adsorption and interaction at liquid/solid interfaces and to relate continuously the different observed effects to solid surface wettability. In this study the gradient method was applied to the analysis of the surface energy dependence of the adsorption of human fibrinogen, γ-globulin, and lysozyme on the solid surfaces. Detergent-induced desorption of adsorbed fibrinogen and γ-globulin was studied by incubating the gradient plates in Tween 20 and sodium dodecyl sulfate (SDS). Only small amounts of protein were desorbed by incubation in Tween for contact angles higher than 80°. Desorption with Tween was instead maximal at about 70° for fibrinogen. The maximum of Tween-induced desorption was shifted to the hydrophilic side of the gradient for γ-globulin. Tween 20 had very little desorption effect at contact angles lower than 30°. SDS caused effective desorption of fibrinogen and γ-globulin on both the hydrophobic and the hydrophilic sides of the gradient. The desorption induced by addition of 4 M urea and acid buffer (pH 2.3) was also studied and was shown to be maximal at the hydrophilic side of the gradients although there was a considerable amount of protein also desorbed at hydrophobic parts of the gradient. There were other qualitative differences in the desorption pattern of γ-globulin and fibrinogen which may be partly explained by assuming different degrees of surface-induced conformational changes of the adsorbed protein molecules.

Malakoplakia: Evidence for Monocyte Lysosomal Abnormality Correctable by Cholinergic Agonist in Vitro and in Vivo

We studied monocyte function in a case of malakoplakia in an attempt to characterize the immune defect in this condition. Our patients intracellular cyclic-GMP levels were abnormally low (mean +/- S.D. of 0.17 +/- 0.05 pmol per 10(7) malakoplakia cells, versus 0.79 +/- 0.12 in normals) p less than 0.001). After phagocytosis, his monocytes failed to release beta-glucuronidase. In the bactericidal assay, incubation of the patients monocytes with Escherichia coli allowed growth of 542 +/- 46 colonies, normal monocytes allowed 95 +/- 22 (p less than 0.001). The percentage of monocytes with large lysosomal granules was 23 +/- 4 in the patient and 4 +/- 2 in normal controls. After in vitro incubation of the patients cells or in vivo treatment with bethanechol chloride, the cyclic-GMP levels, bactericidal ability and lysosomal granules of the cells returned to normal levels. Low levels of cyclic-GMP could impair lysosomal function and bacterial killing in this condition. Cholinergic agonists correct the in vitro abnormalities and are beneficial in vivo.

C3 Adsorbed to a Polymer Surface Can Form an Initiating Alternative Pathway Convertase

Contact between blood and a biomaterial surface induces an immediate complement-mediated inflammatory response. Under these conditions, the alternative pathway of complement is often initiated and amplified on the biomaterial surface. Adsorption of a protein such as C3 to a polymer surface induces conformational changes in the protein. Based on the expression on adsorbed C3 of conformational neoepitopes specific for bound C3 fragments, we have hypothesized that adsorbed C3 is able to bind factor B and form a functional C3,Bb convertase. Using a quartz crystal microbalance to monitor binding of proteins to a polymer surface, we have demonstrated that a functional C3-containing alternative pathway convertase can be formed, in particular, in the presence of properdin. These data indicate that adsorption of C3 induces conformational changes that turn C3 into a C3b-like molecule that is able to participate in the functioning of the alternative convertase, and they suggest a new mechanism for complement activation on a biomaterial surface.

Conformational changes of a model protein (complement factor 3) adsorbed on hydrophilic and hydrophobic solid surfaces

Abstract We have used hydrophobic and hydrophilic silicon surfaces as model surfaces to study the importance of solid surface wettability for the conformation of adsorbed C3 molecules. Adsorption of C3 and its subsequent interaction with antibodies were measured by enzyme-linked immunosorbent assay and ellipsometry. Immunochemical evidence for conformational changes of the adsorbed C3 molecule was investigated by the use of antibodies directed against epitopes hidden in the native molecule. It was found that C3 adsorbed on hydrophobic silicon had a conformation exposing antigenic epitopes which are only accessible in C3 denatured by SDS or C3 that have been biologically activated.

Complement activation during cardiopulmonary bypass: Effects of immobilized heparin

The role of complement in biocompatibility reactions and the correlation between complement activation during cardiopulmonary bypass (CPB) and postperfusion syndrome have inspired attempts to improve the biocompatibility of extracorporeal blood oxygenation devices. Here we assessed the effect of immobilized heparin on the generation of C3a and terminal complement complexes during CPB. Thirty patients undergoing aortocoronary bypass were randomized to CPB with either heparin-coated (Duraflo II; Bentley, Irvine, CA) or noncoated control membrane oxygenators (Univox; Bentley). A standard dose of heparin (300 IU/kg) was given to the control group while the heparin dose was reduced to 30% (100 IU/kg) in the heparin-coated group. Significantly lower levels of terminal complement complexes were detected in the heparin-coated group by the end of CPB. From 28 +/- 5 AU/mL (heparin-coated group) and 26 +/- 3 AU/mL (control group, mean +/- standard error of the mean) the terminal complement complex levels increased to 391 +/- 35 AU/mL and 602 +/- 47 AU/mL, respectively (p < 0.002). This difference was still apparent 180 minutes after CPB. Although there was no difference in C3a levels between the two groups at the end of CPB, C3a levels were significantly lower in the heparin-coated group 30 minutes after CPB (194 +/- 18 ng/mL and 307 +/- 18 ng/mL in heparin-coated and control groups, respectively; p < 0.001). We conclude that the heparin-coated surface is more biocompatible with regard to complement activation than is the ordinary unmodified surface in extracorporeal circuits.

Serum immunocomplexes in patients with subarachnoid hemorrhage

Immunocomplexes (IC) in serum were analyzed in 54 patients with subarachnoid hemorrhage (SAH) from ruptured arterial aneurysms. A previous study had shown that patients with SAH and vasospasm had a significantly higher incidence of ICs in the blood than patients without vasospasm. The aim of the present study was to study how the IC content varied with time and compare this pattern with the clinical picture. Forty-two patients presented clinical or radiological signs of cerebral vasospasm during their hospital stays, whereas 12 patients showed no such signs. The patients with vasospasm had a significantly higher amount of ICs in serum than those without vasospasm. In 37 patients with vasospasm, the changes of IC content during the 1st weeks after SAH correlated well with the clinical course. Data indicated that a high IC content preceded the onset of vasospasm and a low content preceded clinical improvement. This observation supports the idea that the presence of ICs might be the cause and not the result of vasospasm.

Complement C3b interactions studied with surface plasmon resonance technique

The surface plasmon resonance (SPR) phenomenon is utilized in a number of new real time biosensors. In this study, we have used this technique to study interactions between the central complement component C3b and its multiple ligands by using the Biacore equipment. The SPR technique is particularly suitable for analysis of the alternative complement pathway (AP) because the inherent nature of the latter is to amplify deposition of C3b on various surfaces. C3b was coupled onto the sensor surface and the coupling efficiency was compared under various conditions on both polystyrene and carboxymethylated dextran surfaces. After enzymatic C3b coupling or standard amine C3b coupling, we analyzed and compared the binding of four C3b ligands to the surface: factor B, factor H, C5 and the soluble complement receptor 1 (sCR1, CD35). Binding of each ligand to C3b was detected when C3b had been coupled either enzymatically or using the amine coupling, but the half-lives of the interactions were found to vary depending on the coupling procedure. Factor H binds to C3b via three interaction sites. The target sites are exposed on the C3b, C3c and C3d fragments of C3, respectively. Therefore, we also tested by using the Biacore whether factor B, C5 and sCR1 bind to C3c and/or C3d. It was found that factor B bound to C3d, but not to C3c. On the other hand, both C5 and sCR1 bound to C3c, but not to C3d. In conclusion, this study shows that SPR is a powerful tool in analyzing and mapping the interactions of C3b with its multiple ligands.

Inhibition of complement activation by soluble recombinant CR1 under conditions resembling those in a cardiopulmonary circuit: reduced up-regulation of CD11b and complete abrogation of binding of PMNs to the biomaterial surface

The influence of soluble recombinant CR1 (sCR1) on complement activation, and its indirect effects on the coagulation system and cellular responses were assessed in two models for the study of blood/surface and blood/air interactions, as are encountered in e.g. cardiopulmonary bypass circuits. The concentrations of C3a and sC5b-9 and the amount of bound C3/C3 fragments were analyzed as indicators of complement activation. Thrombin-antithrombin complexes, the platelet count, surface-ATP, beta-thromboglobulin, and the expression of CD11b on leukocytes were the parameters analyzed to reflect coagulation and cellular responses. In addition, immunochemical analyses of the phenotypes of surface-bound leukocytes and platelets were performed. Recombinant sCR1, at doses ranging between 0.1-0.25 mg/ml, was found to completely inhibit the generation of sC5b-9, and of C3a by two thirds; the binding of C3 and/or C3 fragments to the surface was almost entirely abolished. As a result of the inhibition of complement activation, the expression of CD11b on PMNs, and the binding of these cells to the biomaterial surface was almost completely lost. In contrast, the thrombin-antithrombin complexes, the platelet count, and the adherence of platelets to the surface, as reflected by the ATP binding and the release of beta-thromboglobulin, were not affected. These data show that complement activation, in association with extra-corporeal treatment, causes activation and binding of PMNs to the biomaterial and that these effects can be completely abolished by the addition of soluble recombinant sCR1.

Evidence for iC3 generation during cardiopulmonary bypass as the result of blood-gas interaction

Earlier we have shown that iC3 is generated at the blood‐gas interface in vitro and that the generation of this molecule is independent of complement activation and the composition of the gas. In order to investigate whether iC3 is also generated during cardiopulmonary bypass where blood comes into contact with oxygen bubbles, two bubble oxygenators were incubated at 37°C with human heparinized blood. A continuous increase in the level of iC3 was shown in the oxygen‐perfused bubble oxygenator (up to 100 nmol/l after 180 min) in contrast to the unbubbled control. Similarly, in plasma drawn from patients undergoing cardiopulmonary bypass using either bubble or membrane oxygenators. the levels of iC3 were shown to increase continuously during the operation. Furthermore, this form of C3 was found to be susceptible to cleavage by factor I. The formation of iC3 at the blood‐gas interface in vivo could be a mechanism by which gas bubbles induce clinical manifestations associated with complement activation, e.g. during cardiopulmonary bypass, adult respiratory distress syndrome and decompression sickness.

Transfer of prostasomal CD59 to CD59-deficient red blood cells results in protection against complement-mediated hemolysis.

PROBLEM: Prostasomes isolated from human seminal plasma have complement regulatory properties because of their content of CD59, a glycosylphosphatidylinositol (GPI)‐anchored protein. We investigated a functional role of prostasomes by the possibility of transferring CD59 from prostasomes to rabbit erythrocytes (RE) and human erythrocytes obtained from patients with paroxysmal nocturnal hemoglobinuria (PNH), both types of cells lacking CD59.