Plasma Complement Activation Research Articles

Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood

Samples of olyhydroxyalkanoates (PHAs), polyhydroxybutyrate (PHB) and copolymers poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV) with 4 and 18 mol% hydroxyvalerate, synthesized by the bacteria Ralstonia eutropha B5786, were investigated. PHA films in contact with blood did not activate the hemostasis system at the level of cell response, but they did activate the coagulation system and the complement reaction. To detect biologically-active components in the PHAs, a detailed analysis of the composition of the polymers was conducted. Gas chromatography-mass spectrometry revealed long-chain fatty acids (FAs) in the tested PHAs. Their total concentration in the polymer ranged from tenths of mol% to 2-3 mol%, depending on the purification method. C16:0 constituted the largest proportion, up to 70%. Of the long-chain hydroxy acids, only β-OH-C14:0 was detected and it did not exceed 0.06 mol%. The analysis of the hemocompatibility properties of the PHAs purified by a specialized procedure, including the quantitative and morphological estimation of platelets adherent to the surface of polymer films, the plasma recalcification time and complement activation studies, indicated that PHB and PHBV can be used in contact with blood. It has been found out that the lipopolysaccharides of bacteria producing PHAs, which contain mostly long-chain hydroxy acids, can be the factor activating the hemostasis systems. Thus, the technology of PHA purification must satisfy rather stringent specific requirements.

Coupling complement regulators to immunoglobulin domains generates effective anti-complement reagents with extended half-life in vivo.

Complement activation and subsequent generation of inflammatory molecules and membrane attack complex contributes to the pathology of a number of inflammatory and degenerative diseases, including arthritis, glomerulonephritis and demyelination. Agents that specifically inhibit complement activation might prove beneficial in the treatment of these diseases. Soluble recombinant forms of the naturally occurring membrane complement regulatory proteins (CRP) have been exploited for this purpose. We have undertaken to design better therapeutics based on CRP. Here we describe the generation of soluble, recombinant CRP comprising rat decay accelerating factor (DAF) or rat CD59 expressed as Fc fusion proteins, antibody-like molecules comprising two CRP moieties in place of the antibody Fab arms (CRP-Ig). Reagents bearing DAF on each arm (DAF-Ig), CD59 on each arm (CD59-Ig) and a hybrid reagent containing both DAF and CD59 were generated. All three reagents inhibited C activation in vitro. Compared with soluble CRP lacking Fc domains, activity was reduced, but was fully restored by enzymatic release of the regulator from the Ig moiety, implicating steric constraints in reducing functional activity. In vivo studies showed that DAF-Ig, when compared to soluble DAF, had a much extended half-life in the circulation in rats and concomitantly caused a sustained reduction in plasma complement activity. When given intra-articularly to rats in a model of arthritis, DAF-Ig significantly reduced severity of disease. The data demonstrate the potential of CRP-Ig as reagents for sustained therapy of inflammatory disorders, including arthritis, but emphasize the need for careful design of fusion proteins to retain function.

Activation of the complement system as an indicator of pyrogenic reaction to lipopolysaccharide (LPS)

The generation of biologically active complement split products through the direct reaction of microorganisms with complement proteins is one of the earliest events of the defence reaction in humans. Complement activation develops within minutes, which highly corresponds with the onset of a febrile reaction after exposure to pyrogens. The possibility of the use of complement activation in human plasma as an indicator of pyrogen contamination has been tested. Additionally, the co-stimulatory effect of complement activation on tumor necrosis factor-alpha (TNF-α) production by blood-separated macrophages exposed to lipopolysaccharide (LPS) has been demonstrated. As an indicator of complement activation in test samples, the concentration of the iC3b fragment was measured by using an ELISA system based on neoantigen formation. The 3-h exposure time has been identified as optimal for the test. The variability between iC3b concentrations in untreated control samples obtained from seven unrelated healthy donors was less than 10%, while after activation by 100 ng/ml LPS, it increased to 13%. The lower detection limit has been identified as 10 pg/ml LPS. As the complement test is not affected by drug–cell interactions or cell viability, the test can be used in situations where tested formulations contain active substances, which interfere with a cell-based test. We conclude that a test based on the detection of complement activation in human plasma should be considered as a valuable element of an in vitro pyrogenicity testing battery along with a cell-based assay.

Immune response and disease resistance in Atlantic salmon (Salmo salar L.) fed three levels of dietary vitamin E and the effect of vaccination on the liver status of antioxidant vitamins

Atlantic salmon were fed 40, 300 or 1100 mgall-rac-α-tocopheryl acetate kg feed−1 for 12 weeks.After 6 weeks half of the fish in each group were marked and vaccinated (i.p.injection) against furunculosis and vibriosis. Liver α-tocopherol levelsreflected the dietary input after 6 and 12 weeks of feeding. Noimmunomodulatoryeffects of dietary vitamin E on baseline levels of the immune parameters beforevaccination were detected in this study, as evaluated by antibody dependent andspontaneous plasma complement activities. In general, vaccination increasedplasma complement activities and the number of antigen specific antibodyproducing cells as compared with unvaccinated control fish, but with nodifferences with respect to the vitamin E regimes. Also the ability ofunvaccinated fish to withstand experimental furunculosis was unaffected bydietary vitamin E. The concentrations of α-tocopherol and ascorbic acidinthe liver were, however, negatively affected 6 weeks post vaccination comparedto respective unvaccinated fish.

Effect of long-term oral administration of an immunostimulant diet on innate immunity in sea bass (Dicentrarchus labrax).

Immunostimulants represent a modern and promising tool in aquaculture, enhancing the resistance of cultured fish to disease and stress. This study investigated the effect of a combination of dietary glucans, alpha-tocopherol and ascorbic acid on the innate immune response of cultured sea bass (Dicentrarchus labrax). After 5 weeks of adaptation on a commercial diet containing 100 p.p.m. ascorbic acid and 200 p.p.m. alpha-tocopherol, sea bass were switched to a diet supplemented with 2% beta-1.3/beta-1.6 glucans and ascorbic acid and alpha-tocopherol at 500 p.p.m. The supplemented diet was given at 2% of body weight per day over a 2-week period, every 3 months. Plasma lysozyme concentration, content and distribution of major plasma proteins and complement activity were measured prior to feeding the supplemented diet and after 40 weeks. Alternative pathways of complement activation and lysozyme activity were both significantly enhanced in fish fed on glucans and elevated doses of vitamins. No significant differences were observed in protein content or in albumin/globulin ratio. Compared to lysozyme activity, which showed marked individual variation, complement-mediated haemolytic activity has been shown to be a more reliable indicator of sea bass immunocompetence. Further studies are in progress to clarify the effect of each dietary component on the innate immune response and disease resistance.

Role of tissue factor-mediated coagulation in ischemia/ reperfusion-induced injury of Langendorf-perfused rabbit hearts.

Production of oxygen free radicals, and activation of neutrophils and plasma complement contribute to myocardial reperfusion injury, but the role of coagulation has not been assessed. To characterize tissue-factor-mediated generation of thrombin and its association with tissue injury during reperfusion from normothermic ischemia of isolated, Langendorf-perfused rabbit hearts. Activation of coagulation was assessed by addition of 12% rabbit plasma and human fibrinogen to Krebs-Henseleit-buffer perfusate with measurement of levels of human fibrinopeptide A (hFPA) in the heart effluent as an index of thrombin-mediated formation of fibrin. Concentrations of hFPA in the effluent were minimal during non-ischemic perfusion (5 +/- 5 ng/ml, n=6) and during 50 min of ischemia (13 +/- 3 ng/ml, n=6), but increased markedly during the first 20 min of reperfusion (to 41 +/- 29 ng/ml, P=0.03 versus before reperfusion). Addition to the perfusate of 10 microg/ml recombinant human tissue-factor-pathway inhibitor, the physiologic inhibitor of tissue-factor-mediated coagulation, abolished increases in the level of hFPA after reperfusion. However, indexes of myocardial injury manifested during reperfusion, including decrease in recovery of left ventricular pressure developed, increase in left ventricular end-diastolic pressure, and increase in activity of creatine kinase in the heart effluent, were not improved by anticoagulation with recombinant human tissue-factor-pathway inhibitor. Our results do not support the hypothesis that coagulation plays a major role in ischemia/reperfusion injury of Langendorf-perfused rabbit hearts.

Inflammatory cell recruitment, distribution, and chemiluminescence response at IgG precoated- and thiol functionalized gold surfaces.

The role of complement activation by artificial surfaces relative to inflammatory response is not well understood. This study was performed to evaluate the inflammatory cell recruitment, distribution, and ex vivo metabolic activation of surfaces with different plasma protein adsorption and complement activation properties in vitro. The implants were (1) pure gold (reference), (2) albumin-precoated (3) IgG-precoated gold, and (4) 3-mercapto-1, 2-propanediol [mercaptoglycerol (MG)] and (5) glutathione (GSH) immobilized to gold. The implant disks were inserted subcutaneously in rats for 24 h, and the number of inflammatory cells that were recruited to the implant adjacent to the surrounding fluid phase (exudate) and the surfaces were quantified by DNA measurements. The oxidative burst was analyzed ex vivo using spontaneous and phorbol myristate acetate (PMA)-stimulated, luminol-enhanced chemiluminescence (CL). The in vitro surface-induced anti-rat C3 binding was evaluated by ellipsometry and antibody techniques after plasma incubations for 1 and 30 min. The ellipsometric results showed that immobilized mercaptoglycerol and IgG-coated, but not the immobilized glutathione or the reference Au, bound anti-C3. The in vivo results revealed that the largest amount of cells was associated with the IgG-coated surfaces, followed by immobilized GSH and MG, albumin-coated, and gold surfaces, respectively. No spontaneous ex vivo luminol-enhanced CL was recorded from the cells irrespective of surface functionality or localization. A down-regulation of surface-associated and exudate leukocyte CL was observed ex vivo, irrespective of surface functionality. The results do not indicate a clear relationship between the degree of complement activation in vitro and leukocyte recruitment and adhesion in vivo for differently functionalized surfaces.

Possible Mechanism for in Vitro Complement Activation in Blood and Plasma Samples: Futhan/EDTA Controls in Vitro Complement Activation

Ongoing in vitro complement (C) activation in citrate or EDTA plasma has prevented an accurate analysis of C-activation products generated in vivo. The aim of this study was to characterize handling and storage conditions required to prevent in vitro C activation in blood and plasma samples collected with Futhan/EDTA. Biotrak(TM) RIAs were used to quantitatively measure C3a and C4a in blood and/or plasma samples from healthy individuals (controls) and from liver transplant patients. Blood samples were routinely drawn into either EDTA (1 g/L) tubes or into tubes containing both EDTA (1 g/L) and Futhan (0.1 g/L) and immediately centrifuged at 2000g for 15 min at 4 degrees C. In controls, C4a, but not C3a, in fresh samples (time 0) was higher in EDTA plasma than in Futhan/EDTA plasma (n = 20; P = 0.002). Futhan/EDTA prevented C3a and C4a generation in blood and plasma samples held at room temperature (22-23 degrees C) for 1 h and in plasma held for 24 h at 4 degrees C or -70 degrees C. The mean C3a concentration (1.76 mg/L; n = 19) at time 0 in EDTA plasma samples from liver transplant patients was significantly higher than for controls (0.34 mg/L; n = 11). In these patients, the mean C3a in EDTA samples increased to 13.8 mg/L after 60 min at room temperature, but there was no change in the C3a concentration of an EDTA plasma from a control. In the patients, C3a concentrations were lower in Futhan/EDTA plasma than in EDTA at time 0 and after 60 min at room temperature (1.40 and 2.02 mg/L, respectively). The mean patient C4a was 4.02 mg/L in EDTA plasma at time 0 vs 0.24 mg/L for controls; it increased to 16.9 mg/L after 60 min at room temperature compared with 0.76 mg/L for controls. The mean patient C4a was 0.83 mg/L in Futhan/EDTA plasma at time 0 vs 0.1 mg/L for controls. Neither patient nor control C4a concentrations increased vs time in Futhan/EDTA. The combination of Futhan (0.1 g/L) and EDTA (1 g/L) eliminates in vitro C activation.

White blood cell counts and plasma C3a have synergistic predictive value in patients at risk for acute respiratory distress syndrome

To investigate and select nonassociated variables with predictive value for acute respiratory distress syndrome (ARDS) in patients at risk. Prospective, observational study. A university hospital intensive care unit. Twenty-four critically ill patients with different risk factors for ARDS. Arterial and mixed venous blood, as well as urine samples, were collected. Invasive hemodynamic measurements were performed. Fifty-nine variables pertaining to the cardiorespiratory, hepatic, immunologic, and renal systems and including plasma complement activation products C3a and SC5b-9 and polymorphonuclear elastase, were determined every 6 hrs for 3 days in patients at risk for ARDS. Associations among variables were investigated and the predictive value of nonassociated variables for ARDS was determined. Patients who developed ARDS (n=8) had lower white blood cell counts at the time they entered the study (p=.006) and during the first 24 hrs thereafter (p=.032). Also, plasma C3a concentrations were markedly higher during the first 24 hrs in patients who developed ARDS (p=.006). Plasma C3a had better predictive value than did white blood cell counts for cutoff points set by discriminant analysis at 1075 ng/mL (1.075 x 10(-3) g/L) and 5700 cells/mL, respectively. The combination of both variables in a discriminant function improved the predictive value for ARDS. The most notable and nonassociated alterations observed in patients who developed ARDS were lower white blood cell counts and higher plasma C3a concentrations compared with counts and concentrations in patients who did not develop ARDS. Plasma C3a concentrations showed better predictive value than white blood cell counts. The combination of white blood cell counts with plasma C3a concentrations synergistically improved the predictive value for ARDS. This combination may prove useful for identifying subpopulations at highest risk for ARDS and may contribute to make treatment at an early stage of the syndrome possible.

Complement Activation in SeverePlasmodium falciparumMalaria

We determined indices of plasma complement activation (C3, C4, Bb, C4d, iC3b, and SC5b-9), levels of tumor necrosis factor (TNF) and interleukin-6, and the APACHE II score in 23 patients with complicatedPlasmodium falciparummalaria. On admission, plasma concentrations of Bb, SC5b-9, and C4d were markedly increased compared to healthy control subjects (n= 24) (4.5 ± 1.9 vs 1.5 ± 0.6 mg/L; 1125.7 ± 496.9 vs 183.2 ± 76.5 μg/L; and 15.7 ± 5.7 vs 7.2 ± 1.4 mg/L,P< 0.01 for all). In contrast C3 and iC3b concentrations were decreased (631.4 ± 247 vs 947.3 ± 243.2 and 105 ± 17.9 vs 151.3 ± 14.5 mg/L;P< 0.01 for both). Plasma C4 concentrations in malaria were not different from normal controls. Plasma Bb, C3, and iC3b levels normalized on day 7 of treatment, whereas SC5b-9 and C4d levels remained elevated. A significant correlation between elevated TNF levels and Bb (r= 0.507) and SC5b-9 (r= 0.448,P< 0.01 for both) and a negative correlation between iC3b and SC5b-9 and TNF levels existed (r= −0.537 andr= −0.466,P< 0.01 for both). In addition, a significant correlation between C3 and iC3b (r= 0.689) and C4 and C4d (r= 0.737) existed. However, no relation between clinical disease severity and complement fragments existed. The results demonstrate that both the classical and the alternative pathways of the complement system are profoundly activated in complicated malaria.

Potentiation of C1 inhibitor by glycosaminoglycans: dextran sulfate species are effective inhibitors of in vitro complement activation in plasma.

Activation of the complement system may contribute to the pathogenesis of many diseases. Hence, an effective inhibitor of complement might be useful to reduce tissue damage. Some glycosaminoglycans (GAG), such as heparin, are known to inhibit the interaction of C1q with activators and the assembly of the classical and the alternative pathway C3 convertases. Furthermore, they may potentiate C1 inhibitor-mediated inactivation of C1s. To search for potential complement inhibitors, we systematically investigated the complement inhibitory properties of various synthetic and naturally occurring GAG (dextran sulfates 500,000 and 5,000, heparin, N-acetylheparin, heparan sulfate, dermatan sulfate, and chondroitin sulfates A and C). First, we assessed the effect of GAG on the second-order rate constant of the inactivation of C1s by C1 inhibitor. This rate constant increased 6- to 130-fold in the presence of the GAG, dextran sulfate being the most effective. Second, all tested GAG were found to reduce deposition of C4 and C3 on immobilized aggregated human IgG (AHG) and to reduce fluid phase formation of C4b/c and C3b/c in recalcified plasma upon incubation with AHG. Dextran sulfate again was found to be most effective. We conclude that GAG modulate complement activation in vitro and that the low molecular weight dextran sulfate (m.w. 5000) may be a candidate for pharmacologic manipulation of complement activation via potentiation of C1 inhibitor.

Immobilized heparin inhibits the increase in leukocyte surface expression of adhesion molecules.

The effect of heparin coating of artificial materials on the surface expression of leukocyte surface molecules was assessed in blood from 9 donors in an experimental model of extracorporeal circulation. The leukocyte surface molecules CD11b, complement receptor type 3 (CR3), and CD45, leukocyte common antigen (LCA), on granulocytes and monocytes exposed to unmodified polyvinyl chloride tubes increased significantly compared with unmanipulated controls (p < 0.001) whereas a very modest and nonsignificant increase was observed in blood circulated in heparin-coated tubes. Accordingly, a highly significant reduction in expression of the 2 molecules was achieved by heparin coating (p < 0.001). The expression of CD11b and CD45 correlated with the degree of complement activation in plasma (rho = 0.6, p = 0.003). The expression of CD14 (endotoxin receptor) and CD16 (Fc gamma III receptor) was increased to the same extent in blood from unmodified and heparin-coated tubes (p < 0.02), and no correlation to complement activation was seen (rho = -0.275, p = 0.17 and rho = -0.004, p = 1, respectively). In conclusion, heparin coating of artificial surfaces has a substantial effect, reducing the expression of molecules involved in cellular adhesion and activation (CD11b and CD45), and the increase of these molecules by unmodified surfaces is complement dependent. In contrast, up-regulation of other surface molecules (CD14 and CD16) seems to be independent of heparin coating and complement activation and, thus, might be regulated by other mechanisms.

Biomaterials and Granulomas

The rapidly expanding use of implants for reconstruction and repair of diseased or traumatized tissues and organs has fueled the search for biomaterials that are stable, nontoxic, and biologically inert. Unfortunately, implants frequently cause acute or chronic inflammation resulting in tissue damage and rejection. Inflammation usually occurs at the biomaterial–tissue interface and reflects surface adsorption of plasma proteins, complement activation, neutrophil and macrophage infiltration, hyperplasia, and release of inflammatory mediators and proteolytic enzymes. If the biomaterial degrades, either spontaneously or due to biologic activity, components can leach into surrounding tissues and may enter the circulation, causing toxic effects systemically and in distant sites. Inflammation of connective tissues in genetically susceptible individuals can potentially activate the immune system and break tolerance to tissue-specific antigens. Experimentally induced animal models of arthritis chronicle the evolution of a chronic, destructive inflammation localized to peripheral joints. Models of autoimmune arthritis involve hyperimmunization with heterologous cartilage components, such as type II collagen or proteoglycan, leading to cross-reactivity with self-cartilage. Other apparently nonimmune models, oil-induced arthritis and pristane arthritis, may reflect granuloma formation and uncontrolled cytokine production. The antigen-induced arthritis model, developed by hyperimmunization with a foreign antigen that is subsequently injected into the joint cavity, is ideal for biocompatibility testing of cationic substances that may be immunogenic. This variety of models offers the means to understand adverse implant–tissue interactions and granuloma formation such that safer biomaterials can be developed in the future.

Lack of plasma complement activation in severe acute alcoholic hepatitis.

To investigate whether complement pathway activation contributes to the clinical and histological features of acute alcoholic hepatitis, we studied the activation of the classical and alternative pathways in patients with alcoholic hepatitis (n = 20), inactive alcoholic cirrhosis (n = 8), heavy drinkers without alcoholic liver disease (n = 10), patients with liver disease of nonalcoholic etiology (n = 11), and healthy control subjects (n = 18). Complement activation was evaluated in the alcoholic hepatitis patients by its correlation with a number of clinical and laboratory features indicative of the severity of liver injury, as well as by comparison of the patient groups. There was no significant difference in circulating C3 [1.02 g/liter, confidence interval (CI) = 0.76-1.28] or C4 (0.25 g/liter, CI = 0.17-0.33) in patients with alcoholic hepatitis when compared with the four control groups. Factor B levels (0.24 g/liter, CI = 0.21-0.27) were higher in the alcoholic hepatitis patients than the control groups (p < 0.01). However, activation of complement (given by the ratios C3d/C3, C4d/C4, and Ba/factor B) was not different in alcoholic hepatitis patients when compared with the control groups. Univariate analysis of a wide range of clinical and laboratory features in the alcoholic hepatitis subjects showed a positive correlation between plasma C3 and serum alkaline phosphatase (r = 0.68, p = 0.0014), AST (r = 0.55, p = 0.015), and gamma-glutamyltranspeptidase (r = 0.47, p = 0.035), but no correlation with clinical or laboratory features associated with high morbidity or mortality. There is no relationship between clinical or laboratory indicators of disease severity and complement activation, and it is unlikely that complement activation contributes to the clinical and histological features of alcoholic liver disease.

Soluble complement receptor type 1 inhibits complement activation induced by hemodialysis membranes in vitro

A variety of bioincompatible events that occur during hemodialysis have been attributed to complement activation. However, cause-effect relationships have been based primarily on indirect evidence and the results of in vitro studies, because an acceptable method for inhibiting complement activation during clinical hemodialysis has been unavailable. Methods for inactivating complement in vitro are available, but the most commonly used of these techniques (heat inactivation and chelation of divalent cations) lack specificity. A recombinant, soluble form of human complement receptor type 1 (sCR1) has been developed recently and shown to inhibit complement activation in vivo. Here, we report studies aimed at determining the effects of sCR1 on dialysis-induced complement activation and neutrophil degranulation. In a concentration dependent fashion, sCR1 inhibited plasma complement activation by cuprophan membrane in vitro. Using a maximally inhibitory concentration (30 micrograms/ml), sCR1 blocked generation of C3a(desArg) by cuprophan, cellulose acetate, and polymethylmethacrylate membranes by 90%, 84%, and 84%, respectively. In contrast, elastase release (a measure of neutrophil degranulation) was inhibited by 70%, 70%, and 44%, respectively, suggesting that dialysis-induced neutrophil activation is mediated in part by noncomplement dependent mechanisms. Both heat- and EDTA-treatment of plasma abolished dialysis membrane-induced complement activation, but these treatments also affected noncomplement dependent components of the degranulation process. These observations show that, compared with other commonly used methods for inhibiting dialysis induced complement activation, sCR1 is more specific. An additional advantage of sCR1 is its potential for use in the clinical setting.

36 COMPLEMENT ACTIVATION IN THE IDIOPATHIC RESPIRATORY DISTRESS SYNDROME (IRDS)

Complement activation in preterm infants with IRDS may be partly caused by platelet-activating factor (PAF). PAF and complement can activate leukocytes. We measured in plasma complement activation (C3a), PAF-inhibitory capacity (PAF-IC;low PAF-IC indicates PAF release), and leukocyte activation (elastase-proteinase inhibitor complex (E-PI) and tumor necrosis factor (TNF-α)) together with the leukocyte count on day 1, 3 and 5 in preterm infants with IRDS and in healthy 1-day-old preterm infants (reference).Conclusion: The leukocyte count and plasma concentrations of E-PI and TNF-α are reduced in the IRDS infants. This may represent activation and sequestration of leukocytes in the lungs due to PAF and activated complement.

Anaphylatoxin generation and distribution during in vitro LDL apheresis.

The extent of anti-apo-B IgG-Sepharose-induced complement activation in serum and plasma (heparin 2 U/ml and ACD-B 1:20) was investigated using an in vitro model of LDL apheresis. The total volume of serum or plasma loaded to the chromatography column was collected in defined aliquots. The washing, desorption and regeneration fluids were processed in the same way. From the obtained values of generated complement split products C3a (desarg), C4a (desarg), C5a (desarg) and complement proteins C3, C4, C5, the conversion rates of the precursor were calculated. In the experiments with serum, 19% of C3, 8% of C4 and 2.3% of C5 were converted by the immunoadsorbent, whereas with plasma 7, 6, and 0.6%, respectively, were found. Furthermore, only 60-74% of total anaphylatoxins were found in the effluent during the loading process. The residual 26-40% was removed from the column with the subsequent washing fluids. Therefore, in the clinical routine, only a reduced part of generated anaphylatoxins will be retransfused to the patient. The fact that C5 is converted to the most limited extent to its biologically active fragment additionally contributes to the understanding of the good clinical tolerability of the LDL apheresis.

Transfusion of type-A and type-B blood to cats

Summary Although nearly all domestic shorthair and longhair cats have type-A blood (&gt; 99%), the frequency of blood type B in various feline breeds ranges from 0 to 59%. All blood-type-B cats have strong natural alloantibodies, predominantly of the IgM class, whereas blood-type-A cats have low alloantibody titers of the IgG and IgM classes. We therefore studied the efficacy and safety of transfusing 20 ml of matched and mismatched 14C-potassium cyanate-labeled blood to cats. In autologous and allogeneic matched transfusions of blood-type-A and type-B cats, the half-life of labeled erythrocytes proved to be similar (29 to 39 days). In contrast, type-B erythrocytes transfused into 5 blood-type-A cats had a mean (± sd) half-life of only 2.1 ± 0.2 days and induced minor transfusion reactions. Half of the type-A blood given to 4 blood-type-B cats was destroyed within minutes to 6 hours (mean ± sd = 1.3 ± 2.3 hours), depending on the alloantibody titer. After 1 day, none of the labeled erythrocytes were detected. Mismatched transfusions in blood-type-B cats caused marked transient reactions including systemic anaphylactic signs (hypotension, bradycardia, apnea, urination, defecation, vomiting, and severe neurologic depression) and hemolytic signs (hemoglobinemia and pigmenturia) associated with severe reduction in plasma alloantibody titer and complement activity. We conclude that the rapid destruction of type-A erythrocytes transfused into blood-type-B cats predominantly occurs intravascularly and is complement- and IgM-mediated, whereas type-B erythrocytes administered to blood-type-A cats are mostly extravascularly hemolysed by a process involving small amounts of IgG and IgM, but without marked complement activation. Thus, any first or subsequent AB-mismatched transfusions are ineffective and life threatening. We therefore recommend the practice of blood typing or cross-matching prior to transfusing blood in cats to prevent any incompatibility reactions and to assure efficacy.

Studies on quantitative levels of complement activation induced by immobilized and soluble forms of protein A: Relevance to extracorporeal immunoadsorption

Soluble staphylococcal protein A (SpA) has been shown to activate the complement system and complement activation has been demonstrated in patient plasma after extracorporeal immunoadsorption through SpA columns. Studies were performed to determine whether complement activation in human plasma is caused by immobilized SpA and/or minute quantities of soluble SpA which may “leach” from immunoadsorbent columns into plasma. Examination of several clinical plasma samples, after immunoadsorption through SpA columns, revealed minute quantities of soluble SpA which “leached” from the immunoadsorbent matrix ranging in concentration from 0.23 to 0.70 μg/mL. Additional studies indicated that the SpA/silica matrix utilized in immunoadsorbent columns activated the complement system and generated anaphylatoxins C3a, C4a and C5a. Moreover, soluble SpA, at concentrations approximating levels detected due to “leaching”, did not generate anaphylatoxins; higher concentrations of soluble SpA were observed to activate the complement system. Staphylococcal enterotoxin B (SEB), a potential contaminant of SpA preparations, did not activate the complement system. These results indicate that the complement activation observed in human plasma after extracorporeal immunoadsorption is due to the immobilized form of SpA rather than the minute quantities of SpA “leached” from the SpA/silica immunoadsorbent matrix.

Lack of Complement Activation in the Seminal Plasma of Men With Antisperm Antibodies Associated In Vivo on Their Sperm

A specific and sensitive "sandwich"-type radiolabeled antiglobulin assay (RAA) using monoclonal anti-human C5b-9 neoantigen and polyclonal anti-human C5b-9 was used to evaluate the presence of the in vivo product of human complement (C) activation (SC5b-9) in the seminal plasma (SP) of 19 fertile and 61 infertile men. SP SC5b-9 was detectable in 7 (8.7%; 1 fertile and 6 infertile men) of the 80 men with a range of 10 to 175 ng/ml. Levels of SP SC5b-9 in other men were below the limit of detection (less than 10 ng/ml). Of the 33 infertile men with sperm-associated immunoglobulin (Ig) G and/or IgA, 27 (82%) had undetectable levels of SP SC5b-9 immunoreactivity. There was no correlation between the SP SC5b-9 levels and the degree of sperm-associated IgG (r = 0.086) or IgA (r = 0.23) activity. However, significant deposition of sperm-bound C5b-9 due to autologous C activation was demonstrated by flow cytometry of donor sperm treated with sera from autoimmune men with ASA in their sera and on their sperm. These findings suggest that sperm-bound Ig cannot activate C in SP.