Kolbjørn Høgåsen

Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused pig xenografts

Fiane AE, Mollnes TE, Videm V, Hovig T, Høgåsen K, Mellbye OJ, Spruce L, Moore WT, Sahu A, Lambris JD. Compstatin, a peptide inhibitor of C3, prolongs survival of ex vivo perfused xenografts. Xenotransplantation 1999; 6: 000‐000 ©Munksgaard, Copenhagen

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.

Centrifugal pump and heparin coating improves cardiopulmonary bypass biocompatibility

BACKGROUND Centrifugal pumps are being used increasingly for short-term extracorporeal circulation purposes such as during heart operations. Whether the centrifugal pump improves the cardiopulmonary bypass biocompatibility has not been fully documented. METHODS A roller pump (n = 20) was compared in vivo with a centrifugal pump (n = 20) in groups of patients in which cardiopulmonary bypass circuits that were either totally heparin coated (Carmeda BioActive Surface; n = 20) or uncoated (n = 20) were used. We expected the heparin coating to attenuate blood activation, thus possibly making the comparison of the two pumps easier with respect to their different blood activation potentials. Samples of blood plasma, obtained during cardiopulmonary bypass from low-risk coronary artery bypass grafting patients, were analyzed for hemolysis (plasma haemoglobin), complement activation (C3bc and the terminal complement complex), a complement lytic inhibitor (vitronectin), coagulation activation (fibrinopeptide A), granulocyte activation (lactoferrin), and platelet activation (beta-thromboglobulin). RESULTS The concentrations of terminal complement complex, lactoferrin, and beta-thromboglobulin were significantly lower in association with heparin-coated surfaces. The concentration of plasma hemoglobin was significantly lower in association with the centrifugal pump. In uncoated circuits, the beta-thromboglobulin level was significantly higher in association with the roller pump than with the centrifugal pump, but this significant reduction in the beta-thromboglobulin level did not hold true for the heparin-coated circuit group. CONCLUSIONS A heparin-coated cardiopulmonary bypass surface reduces the blood activation potential during cardiopulmonary bypass, and the centrifugal pump causes less hemolysis than the roller pump.

The Molecular Basis for Hereditary Porcine Membranoproliferative Glomerulonephritis Type II Point Mutations in the Factor H Coding Sequence Block Protein Secretion

Porcine membranoproliferative glomerulonephritis type II in piglets of the Norwegian Yorkshire breed is considered the first animal model of human dense deposit disease. Porcine dense deposit disease is caused by the absence of the complement regulator factor H in plasma. Here we report the molecular basis for this absence. Single nucleotide exchanges at position C1590G and T3610G in the coding region of the factor H gene result in amino acid exchanges at nonframework residues L493V and I1166R that are located within SCR 9 and SCR 20, respectively. Apparently the L493V mutation represents a polymorphism whereas the I1166R causes the physiological consequences a block in protein secretion. Expression analysis shows comparable mRNA levels for factor H in liver tissue derived from both affected and healthy animals. In affected piglets, factor H protein is detected in increased amounts in liver cells. Factor H accumulates inside the hepatocytes and is not released as shown by Western blot analysis and immunohistochemistry. These data demonstrate that single amino acid exchanges of two nonframework amino acids either alone or in combination block protein secretion of factor H. This observation is also of interest for other human diseases in which factor H is involved, such as human factor H-associated form of hemolytic uremic syndrome.

Inhibition of Complement-Mediated Red Cell Lysis by Immunoglobulins is Dependent on the IG Isotype and its Cl Binding Properties

We have investigated the effect on complement activation of human irnmunoglohulins (Ig) using several therapeutic Ig preparations including two for intravenous use (IVIG), and various purified myeloma proteins. Ig inhibited lysis in a dose‐dependent manner in the classical pathway assay whereas no alternative pathway inhibition was observed. The Fc part of the molecule was responsible for all the inhibitory effect. Purified IgG3 myeloma proteins were potent inhibitors whereas IgGl inhibited to a lesser extent and lgG2 and IgG4 did not inhibit at all. Inhibition was obtained both when Ig was added to the solution and when it was coated onto a solid matrix. Analysis of the soluble and solid phase Ig after incubation revealed binding of Clq and activated C4 and C3 to the isotypes which inhibited lysis. Using selectively depleted sera and reconstitution with their respective purified components, efficient inhibition of lysis was seen when Ig was added prior to serum (C1), some inhibition was seen at the C4 level, whereas no effect was seen when Ig was added at the C9 level. We conclude that the complementmodulatory effect of Fg in vitro is isotype specific and dependent mainly on competitive C1 binding by the Ig molecule in the absence of antigen.

ACTIVATION OF THE COMPLEMENT, COAGULATION, FIBRINOLYTIC AND KALLIKREIN-KININ SYSTEMS DURING ATTACKS OF HEREDITARY ANGIOEDEMA

Five patients with hereditary angioedema (HAE) were studied during attacks and remission as were healthy controls. The high levels of C1/C1‐INH complexes, low C4 and high ratio C4 activation products (C4bc)/C4 also differed significantly during remission compared to controls.During attacks C4bc/C4 increased (922–2007; P=0.022, remission versus attacks, median values throughout), C2 and CH50 dropped (111–31%; P=0.043 and 110–36%; P=0.016, respectively), TCC (C5b‐9) increased (0.88–1.23 AU/ml; P=0.028). Cleavage of HK increased to be almost complete during attacks (20–90%; P=0.009). While factor XIa/serpin‐complexes did not increase, a more than twofold rise in thrombin/antithrombin‐complexes (0.20–0.50 μg/l; P=0.009) and in plasmin/alpha‐2‐antiplasmin‐complexes (7.3–17 nmol/l; P=0.028) was observed. For the first time cascade activation in HAE was studied simultaneously, and corroborates that attacks lead to activation of the kallikrein‐kinin system, fibrinolysis and early part of the classical complement pathway. In addition, the authors present novel data of terminal complement and coagulation activation, the latter apparently not via FXIa.

EFFECT OF WHOLE AND FRACTIONATED INTRAVENOUS IMMUNOGLOBULIN ON COMPLEMENT IN VITRO

Intravenous immunoglobulins (IVIG) are increasingly used for treatment of inflammatory diseases, and the modulation of complement may contribute to some of its beneficial effects. IVIG may bind C1q and activated C3 and C4, and enhance inactivation of C3b. We have previously shown that IVIG inhibited complement-mediated lysis solely via its Fc part through interaction with the classical pathway. In the present study we have investigated whole IVIG (Octagam, and Sandoglobulin) and the monomer, dimer and multimer fractions of Octagam with respect to complement activation in serum and inhibition of complement lysis of red cells. The isolated fractions were found to be stable, homogeneous (monomer, dimer or multimer) and pure (virtually only IgG). Both whole IVIG and its fractions significantly activated complement in serum and inhibited hemolysis compared with human albumin. These effects were most pronounced in the monomer, less in the multimer and least in the dimer fraction. The complement activation was shown to be mediated through the classical pathway since formation of C1rs-C1inh complexes and C4bc were increased, in contrast to Bb. Surprisingly, heat aggregation of Octagam was not followed by a corresponding increase in complement activation, as would be expected, unless it was dialysed before heating, suggesting that it is stabilized to avoid excess activation. In conclusion, the results support the hypothesis that IVIG causes a mild activation of complement in vitro. We suggest that this effect may contribute to the complement inhibitory properties of IVIG by diverting complement deposition from the target to the fluid phase.

Increased systemic activation of neutrophils but not complement in preeclampsia

Objective To investigate whether neutrophils and systemic complement are activated in pregnancies complicated by preeclampsia more than in normal pregnancies. Methods We measured native complement components and activation products in plasma by enzyme immunoassays in 19 women with uncomplicated pregnancies, 15 with preeclampsia before cesarean deliveries, and 16 nonpregnant women. Neutrophil activation was measured by specific enzyme immunoassays for myeloperoxidase and lactoferrin. Results Myeloperoxidase was significantly higher in women with preeclampsia (197 μg/L, 95% confidence interval [CI] 94, 646) than in women with uncomplicated pregnancies (124 μg/L, 95% CI 70, 289; P = .009), whereas lactoferrin did not differ between groups. C4 was decreased in preeclamptic women (0.16 g/L, 95% CI 0.07, 0.48) compared with women with uncomplicated pregnancies (0.21, 95% CI 0.10, 0.30, P < .001). There were no differences for the other native complement components. There was a significant decrease in C1rs-C1 inhibitor, 13 AU/mL (95% CI 9, 34) versus 19 (95% CI 13, 38) (P ≤ .001) in normal pregnant women compared with nonpregnant women. There also was an increase in C3, C4, C9 (data not shown), C4bp, 132% (95% CI 94%, 161%) versus 91% (95% CI 57%, 128%); C3bc (7.4 AU/mL, 95% CI 4.2, 10.7) versus 4.8 AU/mL (95% CI 3.2, 7.3) and C4bc (8.6 AU/mL, 95% CI 5.7, 14.0) versus 3.5 AU/mL (95% CI 2.2, 6.7) in normal pregnant women compared with nonpregnant women (P ≤ .001). Conclusion Neutrophil activation in preeclampsia was shown by systemic increases in myeloperoxidase. Except for a decrease in C4, systemic complement activation could not be detected in preeclampsia.

Quantitation of vitronectin and clusterin: Pitfalls and solutions in enzyme immunoassays for adhesive proteins

Vitronectin (S protein) and clusterin (SP-40,40/cytolysis inhibitor) are non-homologous, multifunctional proteins which both inhibit complement lysis. Vitronectin is an adhesive protein which binds strongly to polystyrene by hydrophobic interactions. The current study demonstrated that clusterin adsorbed even more efficiently to polystyrene than did vitronectin. This adsorption increased in the presence of Tween 20 and was not abolished by blocking or by the use of other detergents. In double antibody enzyme immunoassays such non-specific binding might invalidate the results. However, the non-specific binding of both proteins was efficiently abolished by the following experimental format: Dynatech Immulon 2 microtiter plate, acidic sample buffer (pH 6.0) containing 0.2% Tween 20 and high sample dilution. Vitronectin was successfully quantitated using this approach, but the measurement of clusterin was not reliable because of high inter-well variation of binding. However, since few serum proteins adsorb to polystyrene in the presence of detergents, clusterin was successfully quantitated in a single antibody enzyme immunoassay in which samples were coated directly onto Nunc Maxisorp plates in the presence of 0.2% Tween 20. In normal blood donors the serum concentration (median and 2.5-97.5 percentile) of vitronectin was 0.34 g/l (0.24-0.53) and of clusterin 0.34 g/l (0.25-0.42).

Complement activation and bioincompatibility. The terminal complement complex for evaluation and surface modification with heparin for improvement of biomaterials.

The degree of biocompatibility of biomaterials can be evaluated using various assay systems detecting activation of the blood cascade systems, leukocytes or platelets. Activation of complement is one mechanism associated with adverse effects observed when bioincompatible materials are used. We present data showing that the terminal complement compiex, an indicator of terminal pathway activation, is suitable for evaluation of biocompatibility of biomaterials such as cardiopulmonary bypass devices. Furthermore, our results suggest that bioincompatibility is improved when artificial surfaces are modified with end point attached functionally active heparin.