Anti-TNF treatment of baboons with sepsis reduces TNF-alpha, IL-6 and IL-8, but not the degree of complement activation.

The activation of complement and the release of TNF-alpha, IL-6 and IL-8 are important pathogenic factors behind organ dysfunction in sepsis. The aim of this study was to determine whether infusion of anti-TNF antibodies alters complement activation and plasma concentrations of pro-inflammatory cytokines at high doses of Escherichia coli. Six baboons received intravenously 2 x 10(9) live E. coli bacteria per kg body weight (group 1), in addition five received pretreatment with 1 mg per kg body weight anti-TNF antibodies (group 2), and seven received 5 x 10(8) live E. coli bacteria per kg body weight (group 3). Two hours after the start of infusion of the bacteria, plasma concentrations of C3 activation products, C5a and the terminal SC5b-9 complement complex were increased in groups 1 and 2 (P < 0.05), but there was no significant difference between the groups. At 2 h the levels of TNF-alpha, IL-6 and IL-8 were lower in group 2 compared with group 1 (P<0.05). In group 2 compared with group 1 the TNF-alpha concentrations were, however, higher at 4, 8 and 24 h. The explanation for this phenomenon is probably that TNF-alpha binds to the anti-TNF antibody complex and is released slowly after it has been bound. The study showed that infusion of anti-TNF antibodies reduced the concentrations of TNF-alpha, IL-6 and IL-8, without any detectable influence on complement activation.

Local and systemic reactions after lung contusion: an experimental study in the pig.

The present study was designed to investigate the consequences of isolated unilateral lung contusion on local alveolar and systemic inflammatory responses in an animal model in the pig. Isolated unilateral lung contusion was induced by bolt shot in eight mechanically ventilated animals under general anesthesia (sham: n=4). Plasma and bronchoalveolar lavage fluid were collected during a period of 8 h following lung contusion. Leukocytes, leukocyte neutral protease inhibitor (LNPI), terminal complement complex (TCC), thrombin-antithrombin-complex (TAT) as well as pulmonary microvascular permeability and surfactant function were determined. Within 30 min, lung contusion was found to cause a significant local and systemic increase in TCC and TAT concentrations and a systemic increase in LNPI concentrations. The latter was accompanied by a sequestration of leukocytes in the contused lung. Complement activation and leukocyte sequestration in the contused lung progressively increased during the investigation period. Although surfactant function decreased in the entire lung 30 min after contusion, TCC, TAT, and leukocyte sequestration was unchanged in the contralateral lung. The first indication of an involvement of the contralateral lung was obtained by an increase in leukocyte sequestration 8 h after lung contusion. Unilateral lung contusion initiates an early systemic activation of humoral and cellular defense systems. Involvement of the contralateral lung appears to be a secondary event caused by a systemic inflammatory reaction.

Effect of soluble complement receptor type 1 on reperfusion edema and neutrophil migration after lung allotransplantation in swine.

OBJECTIVE: Soluble complement receptor type 1 inhibits complement activation by blocking C3 and C5 convertases of the classical and alternative pathways. We evaluated the effect of soluble complement receptor type 1 on lung allograft reperfusion injury. METHODS: Left lung transplantation was performed in 13 weight-matched pigs (25 to 31 kg) after prolonged preservation (20 hours at 1 degree C). One hour after reperfusion the recipient contralateral right lung was excluded to assess graft function only. Complement activity and C3a levels were measured after reperfusion and at the end of the assessment. Extravascular lung water index, intrathoracic blood volume, and cardiac output were assessed during a 5-hour observation period. Gas exchange and hemodynamics were monitored. At the end of the 5-hour assessment period, myeloperoxidase assay and bronchoalveolar lavage were performed to assess neutrophil migration, and C5b-9 (membrane attack complex) deposits in the allograft were detected by immunohistochemistry. Two groups were studied. In group II (n = 6) recipient animals were treated with soluble complement receptor type 1 (15 mg/kg) 15 minutes before reperfusion. Group I (n = 7) served as the control group. RESULTS: Serum complement activity was completely inhibited in group II. In contrast to group I, C5b-9 complexes were not detected in group II allograft tissue samples. C3a was reduced to normal levels in group II (p = 0.00005). Extravascular lung water index was higher in group I animals throughout the assessment period (p = 0.035). No significant difference in allograft myeloperoxidase activity (p = 0.10) and polymorphonuclear leukocyte count of the bronchoalveolar lavage fluid (p = 0.057) was detected. CONCLUSION: Inhibition of the complement system by soluble complement receptor type 1 blocks local complement activation in the allograft and reduces posttransplantation reperfusion edema but does not improve hemodynamic parameters.

Effect of filtration and storage of platelet concentrates on the production of the chemotaxins C5a, interleukin 8, tumor necrosis factor alpha, and leukotriene B4.

BACKGROUND: The production in platelet concentrates (PCs) of C3 activation products (C3bc), terminal complement complex (TCC), and chemotaxins C5a, interleukin (IL)-8, tumor necrosis factor alpha (TNFalpha), and leukotriene B4 (LTB4) and the proposed reduction in concentration of the chemotaxins by white cell reduction were examined. STUDY DESIGN AND METHODS: Samples were collected from supernatants of PCs produced by apheresis (apheresis PCs) or from buffy coats (BC PCs) immediately after the production, after white cell-reduction filtration on Day 1, and after 5-day storage, and examined by enzyme immunoassays. RESULTS: Complement was activated in all PCs during storage, and the concentration of activation products was not influenced by prestorage filtration. In prestorage white cell-reduced BC PCs, only C3bc levels increased. Levels of IL-8, TNFalpha, and LTB4 increased during storage of apheresis PCs, but not in filtered units, except for LTB4. In contrast, levels of IL-8 decreased after storage of filtered BC PCs. C5a correlated significantly with IL-8, which also correlated with TNFalpha and LTB4. CONCLUSION: Both C5a and TNFalpha generation in apheresis PCs seem to induce white cell IL-8 production, which mediates cellular LTB4 release. Prestorage white cell reduction is recommended for reducing chemotactic cytokine and leukotriene levels in all PCs. Production of BC PCs is recommended to achieve less complement activation, which is not affected by filtration.

In situ complement activation in porcine membranoproliferative glomerulonephritis type II.

Pigs genetically deficient in complement factor H all develop lethal membranoproliferative glomerulonephritis (MPGN) type II characterized by massive glomerular deposits of complement, intramembranous dense deposits, and mesangial hypercellularity. To elucidate the chronological relationship between these glomerular changes, and to precisely determine the localization of glomerular complement deposits, we studied kidney specimens from factor H-deficient piglets at different ages from fetal life until terminal kidney failure had developed. Deposits of C3 and the terminal complement complex localized within the glomerular basement membrane (GBM) were present already in factor H-deficient fetuses, without concurrent intramembranous dense deposits or mesangial hypercellularity. Incipient subendothelial dense deposits containing complement appeared no earlier than four days after birth, and intramembranous dense deposits in older piglets with established MPGN type II also contained large amounts of complement as detected by immune electron microscopy. Onset of kidney failure coincided with pronounced mesangial hypercellularity and expansion, compromising glomerular capillary patency. Formation of glomerular capillary wall double contours coincided with electron microscopic evidence of laminar disintegration of intramembranous dense deposits. Complement was also deposited in the mesangial matrix, but not on glomerular cells. We conclude that all components of the alternative and terminal pathways of complement have access into the GBM and the mesangial matrix. In the absence of factor H, complement is spontaneously activated and deposited in situ in these locations resulting in dense deposit formation. It is proposed that factor H dysfunction may play an essential role even in human MPGN type II.

Both plasma- and leukocyte-associated C5a are essential for assessment of C5a generation in vivo.

BACKGROUND: Measurement of C5a in plasma is hampered by the rapid clearance of C5a as a result of cell binding. Therefore, an assessment of whether cell-bound C5a might better reflect C5a generation in vivo is essential. METHODS: We quantified plasma and leukocyte-bound C5a in samples from patients undergoing cardiopulmonary bypass, which is known to be associated with complement activation. C3 activation products and the terminal complement complex were measured as well. RESULTS: Plasma levels of C3 activation products and the terminal complement complex increased rapidly and significantly after the onset of cardiopulmonary bypass until they reached a plateau after 30 minutes. The concentration of plasma C5a increased steadily to twice baseline at the end of bypass. The concentration of leukocyte-associated C5a increased threefold after 10 minutes of cardiopulmonary bypass, when a plateau was reached. A positive correlation was found between levels of plasma C3 activation products or terminal complement complex and plasma C5a plus cell-associated C5a but not between C3 activation products or terminal complement complex and either one of the C5a variables. CONCLUSIONS: We conclude that both plasma C5a and leukocyte-associated C5a are needed for monitoring in vivo C5a generation.

Eradication of porcine factor H deficiency in Norway.

In pigs a hereditary deficiency of the complement-inhibitory protein factor H consistently leads to the development of lethal membranoproliferative glomerulonephritis type II. This autosomal recessive disease has been a common cause of early losses of piglets in the Norwegian Yorkshire breed, but has not been reported in the Norwegian Landrace breed. The aim of the present work was to identify carriers of factor H deficiency and to eradicate the disease from commercial pig populations. Factor H in plasma was measured by an enzyme immunoassay. Sixteen known carriers of the disease (parents of factor H-deficient offspring) had half the level of factor H (median 110, range 87 to 156 mg/litre) recorded in 17 homozygous healthy Yorkshire pigs (median 212, range 183 to 293 mg/litre) and 20 Landrace pigs (median 227, range 200 to 255 mg/litre). Factor H analysis in 397 piglets produced by the mating of known carriers revealed an approximately 1:2:1 distribution of individuals with very low, half-normal and normal levels of factor H representing homozygous deficient, heterozygous and homozygous healthy individuals. Thus, carriers could be identified reliably by measuring the plasma concentration of factor H. Most of the population of Norwegian Yorkshire breeding pigs (490 pigs) was therefore examined, and a half-normal factor H level consistent with the carrier state was found in 13.5 per cent. These animals were prevented from breeding and since then no losses of piglets suspected of being due to factor H deficiency have been reported. No carrier was identified among 102 Norwegian Landrace boars, almost excluding the existence of factor H deficiency in this breed.

Infection with human cytomegalovirus (HCMV) stimulates monocyte production of complement factor 3.

Complement biosynthesis in monocytes is stimulated by different microorganisms including Gram negative bacteria and yeasts. We have tested the effect of human cytomegalovirus (HCMV) on complement factor 3 (C3) production by cultured human monocytes. The monocytes were challenged with either a crude or a purified HCMV preparation obtained from the supernatant of HCMV-infected fibroblasts. When the monocytes were infected with 2 pfu/cell of virus and cultured for 2 days, the increase in C3 production compared to control ranged from 3% to 162%, median 62% (p < 0.01). However, crude HCMV was even more potent in stimulating C3 production, as the increase in C3 values ranged from 104% to 507%, median 247% (p = 0.001). This indicates the presence in the crude HCMV preparation of a substance which acts synergistically with HCMV on the C3 production. When monocytes were stimulated by lipopolysaccharide (LPS), a well known inducer of C3, infection with crude or purified HCMV did not further increase C3 production. Both HCMV and substances produced during the propagation of HCMV in fibroblasts are able to stimulate C3 production in monocytes. Complement production by inflammatory cells may be of importance in host resistance against viral infections.

Attenuation of changes in leukocyte surface markers and complement activation with heparin-coated cardiopulmonary bypass.

BACKGROUND: The inflammatory response induced by cardiopulmonary bypass can result in severe organ dysfunction in some patients. This postperfusion response is caused mainly by contact between blood and the foreign surface of the cardiopulmonary bypass equipment and includes adhesion of leukocytes to vascular endothelium, which precedes a series of events that mediate inflammatory damage to tissues. METHODS: Low-risk patients accepted for coronary artery bypass grafting were randomized to operation with the cardiopulmonary bypass surface either completely heparin coated (Duraflo II) or uncoated. There were 12 patients in each group. Blood plasma sampled during cardiopulmonary bypass was analyzed for complement activation (C3bc and terminal SC5b-9 complement complex) and neutrophil activation (lactoferrin and myeloperoxidase). In addition, neutrophils, monocytes, and platelets were counted, and the expression of surface markers on the neutrophils and monocytes (complement receptor [CR] 1, CR3, CR4, and L-selectin) and on the platelets (P-selectin and CD41) was quantified with flow cytometry. RESULTS: Clinical and surgical results were similar in both groups. In the group with the heparin-coated surface, the formation of the terminal SC5b-9 complement complex was significantly reduced, and the counts of circulating leukocytes and platelets were significantly less reduced initially but were higher at the end of cardiopulmonary bypass compared with baseline. Also, the expression of CR1, CR3, and CR4 was significantly less upregulated and the L-selectin, significantly less downregulated on monocytes and neutrophils. CONCLUSIONS: We conclude that heparin coating reduces complement activation and attenuates the leukocyte integrin and selectin response that occurs when uncoated circuits are used.

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.

Serum clusterin and vitronectin in alcoholic cirrhosis.

Clusterin and vitronectin are multifunctional regulatory proteins which both serve as complement lysis inhibitors. Previous data have strongly suggested that serum vitronectin is mainly produced in the liver, whereas the biosynthetic origin for serum clusterin has not been determined. In the present study we aimed to determine the role of the liver in producing these proteins and to evaluate the proteins as possible markers of liver failure. We therefore quantified clusterin and vitronectin in serum from patients suffering from alcoholic liver cirrhosis (n = 83), and in serum-free culture supernatants from the hepatoma cell line HepG2. The median clusterin concentration was 0.20 g/l in cirrhosis and 0.37 g/l in the controls, whereas corresponding vitronectin values were 0.19 and 0.26 g/l, respectively. The concentration of both proteins showed significant correlation (p < 0.0001) with disease severity and with established plasma markers of hepatic synthetic function, such as albumin and prothrombin complex. The clusterin level, but not the vitronectin level, correlated with survival (p = 0.005). The rates of synthesis of clusterin, vitronectin and C3 from HepG2 cells were 0.02, 0.21 and 1.9 micrograms/10(6) cells/24 h, respectively. From the present data we conclude that clusterin (as vitronectin and C3) is mainly produced in the liver and may be a useful marker in the evaluation of severity of liver disease and prognosis of patients with alcoholic cirrhosis.

Effects on complement activation and cytokine (TNF-alpha and IL-8) release of infusion of anti-TNF-antibodies or a xanthine derivative (HWA 138) in septic baboons.

BACKGROUND: Sepsis and septic shock lead to activation of the complement cascade and to the release of pro-inflammatory cytokines. METHODS: The effects of E coli infusion and of infusion of anti-TNF antibodies and a xanthine derivative (HWA 138) on complement activation and cytokine release was evaluated in 17 baboons. All animals received 5 x 10(8) live bacteria per kg body weight. Five animals received only bacteria, five received in addition 0.5 mg per kg body weight of anti-TNF-antibody, and seven received an infusion of 6 mg per kg body weight of HWA 138 in addition to the bacteria. RESULTS: In baboons receiving 5 x 10(8) live E coli per kg body weight increased plasma levels of TCC, TNF-alpha and IL-8 were found. The release of TNF-alpha was lower in the group receiving HWA 138 at 2 h after the infusion. In baboons receiving an infusion of anti-TNF antibody the concentration of IL-8 was lower at 2 and 4 h than in animals receiving just E coli or HWA 138. CONCLUSION: Infusion of anti-TNF-antibody before E coli infusion will decrease the formation of IL-8. Infusion of HWA 138 before the E coli infusion will also inhibit the formation of TNF-alpha.

Disparity in blood activation by two different heparin-coated cardiopulmonary bypass systems.

BACKGROUND: Several studies have indicated reduced "blood activation" in heparin-coated cardiopulmonary bypass systems. The present study compares the effect of two different heparin coatings on different blood activation indices. METHODS: Low-risk patients (n = 40) were randomized to coronary artery bypass grafting using cardiopulmonary bypass with surfaces coated entirely by either the Duraflo II heparin coat or the Carmeda Biological Active Surface, or with identical uncoated equipment. In all cases, a standard systemic heparin dosage was used. Complement activation (C3 activation products C3bc and C3a and formation of fluid phase terminal SC5b-9 complement complex), neutrophil activation (lactoferrin and myeloperoxidase), and lytic inhibitors (vitronectin and clusterin) were quantified during cardiopulmonary bypass and 6 hours postoperatively. RESULTS: Heparin coating by either method reduced the formation of terminal SC5b-9 complement complex and the release of lactoferrin and myeloperoxidase compared with uncoated systems. Lactoferrin and myeloperoxidase levels increased significantly during cardiopulmonary bypass in the Duraflo II group, whereas no significant increase was observed in the Carmeda Biological Active Surface group. The least formation of terminal SC5b-9 complement complex and neutrophil activation was observed with the Maxima Carmeda Biological Active Surface-coated equipment. The vitronectin and clusterin concentrations were significantly less reduced in the Duraflo II compared with the control group. This study underlines the importance of terminal SC5b-9 complement complex as a suitable marker in the evaluation of complement activation during cardiopulmonary bypass. CONCLUSIONS: Both heparin coatings reduce blood activation, probably more so with Carmeda Biological Active Surface than with Duraflo II.

Effect of syringe-assisted liposuction on activation of cascade systems and circulating cells when using the superwet or tumescent technique.

Although liposuction is considered to be a relatively safe procedure, several deaths and nonfatal serious complications such as sepsis, toxic shock syndrome, thromboembolic disease, fat emboli, and adult respiratory distress syndrome have been reported. In the present study, we have investigated a wide variety of components belonging to the coagulation, fibrinolytic, plasma kallikrein-kinin, and complement systems in 22 patients undergoing syringe-assisted liposuction using the superwet or tumescent technique. In spite of a relatively high mean aspirate volume (2,648 ml), only small changes over time well within the normal range were found for the different parameters. In nine randomly selected patients, we also measured interleukin 6 and tumor necrosis factor-alpha. The size of the interleukin-6 peaks was found to be of the same order of magnitude as those measured in patients undergoing hernia repair or percutaneous cholecystectomy but lower than those in patients undergoing open cholecystectomy, breast reduction, or breast reconstruction. Tumor necrosis factor-alpha was not detected in any sample in any of the patients. We conclude that syringe-assisted liposuction with the present aspirate volumes using the superwet or tumescent technique represents a small to moderate surgical trauma without clinical significant activation of the cascade systems.

Inhibition of complement-mediated red cell lysis by immunoglobulins is dependent on the IG isotype and its C1 binding properties.

We have investigated the effect on complement activation of human immunoglobulins (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 IgG1 inhibited to a lesser extent and IgG2 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 C1q 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 complement-modulatory effect of Ig in vitro is isotype specific and dependent mainly on competitive C1 binding by the Ig molecule in the absence of antigen.

Hereditary porcine membranoproliferative glomerulonephritis type II is caused by factor H deficiency.

We have recently described hereditary membranoproliferative glomerulonephritis type II in the pig. All affected animals had excessive complement activation, revealed as low plasma C3, elevated plasma terminal complement complex, and massive deposits of complement in the renal glomeruli, and eventually died of renal failure within 11 wk of birth. The aim of the present study was to investigate the cause of complement activation in this disease. Transfusion of normal porcine plasma to affected piglets inhibited complement activation and increased survival. Plasma was successively fractionated and the complement inhibitory effect of each fraction tested in vivo. A single chain 150-kD protein which showed the same complement inhibitory effect as whole plasma was finally isolated. Immunologic cross-reactivity, functional properties, and NH2-terminal sequence identified the protein as factor H. By Western blotting and enzyme immunoassay, membranoproliferative glomerulonephritis-affected piglets were demonstrated to be subtotally deficient in factor H. At 1 wk of age, median (range) factor H concentration was 1.6 mg/liter (1.1-2.3) in deficient animals (n = 13) and 51 mg/liter (26-98) in healthy littermates (n = 52). Our data show that hereditary porcine membrano-proliferative glomerulonephritis type II is caused by factor H deficiency.

Transforming growth factor beta modulates C3 and factor B biosynthesis and complement receptor 3 expression in cultured human monocytes.

Complement biosynthesis in monocytes is stimulated by different pathogens and modulated by a variety of cytokines, but little is known about the possible effect of transforming growth factor beta (TGF-beta) on this monocyte function. We therefore studied the effect of TGF-beta 1 and TGF-beta 2 on constitutive, lipopolysaccharide (LPS)- and Candida albicans-induced monocyte biosynthesis of complement components C3 and factor B. Under all three conditions, both forms of TGF-beta (20 ng/ml) induced a two- to fourfold increase in C3 concentration in monocyte supernatants harvested after 2 or 5 days of cell culture, an effect that was abrogated by cycloheximide. In contrast, constitutive and pathogen-induced production of factor B was suppressed by TGF-beta. The effects of TGF-beta on complement production were neutralized by a monoclonal anti-TGF-beta antibody. Moreover, TGF-beta suppressed the pathogen-induced release of granulocyte-macrophage colony-stimulating factor and down-regulated the expression of complement receptor 3 (CD11b/CD18), while the expression of CD11a/CD18, a related beta 2 integrin, was unaffected. These novel effects of TGF-beta emphasize the immunomodulatory significance of this cytokine.

Fatal myocardial depression and circulatory collapse associated with complement activation induced by plasma infusion in severe porcine sepsis.

We have previously reported that fresh frozen plasma (FFP) may induce a rapid irreversible shock when repeatedly infused in pigs challenged with Gram-negative sepsis. The aims of the present study were to elucidate the cardiovascular nature of the shock and determine the aetiologic role of tumour necrosis factor (TNF), complement activation and halothane anaesthesia. Three groups of anaesthetized piglets were inoculated with a lethal dose of live E. coli bacteria. Groups I (n = 8) and III (n = 8) were anaesthetized with halothane and group II (n = 8) with ketamine. Animals in groups I and II received repeated infusions of FFP, whereas animals in group III received repeated infusions of 7% albumin. Six animals in group I and four animals in group II died during the first plasma infusion. Survival time was significantly longer in group II (P = 0.04) compared to group I. No animals in group III died during the albumin infusions, and no adverse effects were observed during the infusions. In group I the plasma induced shock was characterized by abruptly falling mean arterial pressure, cardiac index, systemic vascular resistance index and left ventricular contractility. Concomitant increases were recorded in left ventricular filling pressure and central venous pressure. Group II demonstrated a similar, but delayed response. Plasma infusion was associated with a significant increase in terminal complement complex (TCC) (P < 0.03 in group I, P < 0.05 in group II) and depletion of serum ionized calcium. We conclude that FFP may induce fatal myocardial depression and circulatory collapse in severe sepsis. Complement activation may be of aetiologic importance.

Complement-mediated permeabilization of platelets by monoclonal antibodies to CD9: inhibition by leupeptin, and effects on the GP Ib-actin-binding protein system.

Two monoclonal antibodies to CD9 of the IgM and IgG2a categories (FN 52 and FN 99), reproducibly induced platelet alterations in platelet-rich plasma by activation of the complement system with membrane incorporation of the pore-forming C5b-9 complex. The permeabilization could be monitored by measurements of extracellular ATP and observed as a shape change followed by an increase in light transmission in the aggregometer, and was associated with formation of tiny platelet aggregates. This could be accomplished by only minor lysis observed as extracellular lactate dehydrogenase (LDH). When leupeptin was added prior to, or immediately after the antibody, a total inhibition of the platelet alterations could be obtained. When added soon after the shape change, leupeptin had little effect on the liberation of ATP. However, whereas the ability of the platelets to become agglutinated by ristocetin was lost during the complement-mediated platelet alterations, addition of leupeptin immediately after the shape change, prevented this loss. The lost ability of the permeabilized platelets to undergo ristocetin-induced agglutination is not ascribed to degradation of GP Ib as this was relatively little affected in these studies as compared to the actin-binding protein (ABP) which was profoundly degraded. This protein represents a link between GP Ib and the submembraneous cytoskeleton, and the inhibition of its degradation by leupeptin, was clearly demonstrated. Experiments with digitonin-induced permeabilization showed that leupeptin did not inhibit permeabilization as such, but it did prevent the loss of ristocetin-induced agglutination even with this inducer.

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).