Anticomplementary activity of horse IgG and F(ab')2 antivenoms.

Envenomation by poisonous animals is a neglected condition according to the World Health Organization (WHO). Antivenoms are included in the WHO Essential Medicines List. It has been assumed that immunoglobulin G (IgG) antivenoms could activate the complement system through Fc and induce early adverse reactions (EARs). However, data in the literature indicate that F(ab')2 fragments can also activate the complement system. Herein, we show that several batches of IgG and F(ab')2 antivenoms from the Butantan, Vital Brazil, and Clodomiro Picado Institutes activated the complement classical pathway and induced the production of C3a; however, only those antivenoms from Clodomiro Picado generated C5a. Different protein profiles (IgG heavy chain, protein contaminants, and aggregates) were observed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analyses. Our results show that various antivenoms from different producers are able to activate the classical pathway of the complement system and generate anaphylatoxins, and these findings suggest that factors, such as composition, contaminant proteins, and aggregates, may influence the anticomplementary activity of antivenoms in vitro. Therefore, there is a need to further improve antivenom production methods to reduce their anticomplementary activity and potential to cause EARs.

Contaminated heparin associated with adverse clinical events and activation of the contact system.

BACKGROUND: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany. METHODS: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine. RESULTS: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine. CONCLUSIONS: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system.

Reducing the effects of the systemic inflammatory response to cardiopulmonary bypass: can single dose steroids blunt systemic inflammatory response syndrome?

The use of cardiopulmonary bypass (CPB) is associated with the development of a significant systemic inflammatory response syndrome (SIRS) which can affect patient outcomes. Multiple pathways are involved in initiating and maintaining SIRS. We studied whether a single dose of steroids (dexamethasone) after the induction of anesthesia could blunt the SIRS from CPB. A prospective, randomized, double-blinded, placebo control trial of 28 patients (13 study vs. 15 control). The study group received 100 mg of dexamethasone whereas the control group received sterile saline. Inclusion criteria were the following: elective coronary artery bypass grafting, less than 80 years old, normal ejection fraction, no acute myocardial infarction. Serum levels of C3a, interleukin (IL)-6, and plasma norepinephrine (PNE) were measured after intubation, 30 minutes after initiation of CPB, 24 and 72 hours after termination of bypass. The study group demonstrated significantly lower levels of IL-6 (p = 0.0005) at 24 hours and PNE (p = 0.05) at 72 hours post-CPB. There were no differences in the C3a levels between the groups. No infections occurred in either group. A single dose of dexamethasone reduces IL-6 and PNE levels associated with CPB. Despite the significant reductions in IL-6 and PNE, there was no effect on clinical outcomes. Additional studies are needed to demonstrate a clinically significant effect on patient outcomes.

Metformin decreases circulating acylation-stimulating protein levels in polycystic ovary syndrome.

AIM: There are no studies that examine the circulating acylation-stimulating protein (ASP) levels in patients with polycystic ovary syndrome (PCOS). The present study was designed to determine the ASP levels in PCOS and to evaluate the effect of metformin on plasma fasting ASP concentrations. METHODS: Twenty women with PCOS and 20 healthy controls matched for age and body mass index (BMI) were included in the study. We determined ASP and other biochemical parameters before and after treatment. RESULTS: Baseline levels of plasma ASP, complement 3 (C3), waist-to-hip ratio (WHR), homeostasis model assessment-insulin resistance index (HOMA-IR), fasting insulin, triglycerides (TG) and very-low-density lipoprotein cholesterol (VLDL-C) were significantly higher in patients than in controls. After 3 months of metformin treatment, BMI, WHR, ASP, C3, fasting glucose, fasting insulin, HOMA-IR, total cholesterol, TG, VLDL-C and free testosterone decreased significantly, whereas apolipoprotein A-I and high-density lipoprotein cholesterol increased significantly. CONCLUSIONS: The major novel information of the present study is that ASP and C3 values are markedly increased in non-obese patients with PCOS, with a decrease evidenced with metformin treatment.

Restoration of natural IgM production from liver B cells by exogenous IL-18 improves the survival of burn-injured mice infected with Pseudomonas aeruginosa.

Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-gamma production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43(+)CD5(-)CD23(-)B220(dim) cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.

Off-pump CABG reduces complement activation but does not significantly affect peripheral endothelial function: a prospective randomized study.

OBJECTIVE: Cardiac surgery initiates a systemic inflammatory response, which may affect endothelial function. The aim of this study was to investigate if off-pump CABG (OPCAB) reduces the postoperative inflammatory response and affects endothelial function less than conventional on-pump CABG. DESIGN: Fifty-two patients submitted for elective CABG were included in a prospective, randomized study. Twenty-six patients were operated with, and 26 without cardiopulmonary bypass (CPB). Plasma levels of complement (C3a), cytokines (IL-8, TNF-alpha), endothelin-1 and neopterin were measured before and during surgery and 2 and 24 h after surgery. Endothelial function was assessed by forearm plethysmography and acetylcholine infusion in 30 patients 2-4 h after surgery. RESULTS: C3a and neopterin concentrations were significantly higher during and early after surgery in the CPB group while TNF-alpha and IL-8 tended to be higher in the CPB group but the difference did not reach statistical significance. Endothelial function did not differ significantly between the two groups. CONCLUSION: OPCAB reduces complement activation compared with on-pump CABG but does not significantly affect TNF-alpha and IL-8 release or endothelial function.

Improvement of bypass circuit biocompatibility: comparison and combination of heparin-coated circuit and nitric oxide gas infusion.

OBJECTIVES: Nitric oxide (NO) gas infusion to the oxygenator, as well as heparin-coated bypass circuits, have been reported to attenuate blood activation induced by the interaction with the artificial surfaces of an extracorporeal bypass circuit. Using a mock circulation model, we compared the effect of each and also evaluated the effect of their combination on attenuating bypass-induced blood activation. METHODS: A miniature closed bypass circuit was primed with diluted fresh human blood and perfused for 180 minutes using a centrifugal pump. NO gas (0, 50, or 100 ppm) was infused to the oxygenator sweep gas of either a non-heparin-coated or a heparin-coated circuit. Platelet counts, beta-thromboglobulin, platelet factor 4, complement-3 activation products and granulocyte elastase were measured at 0, 30, 60, 120, and 180 minutes after starting the perfusion. RESULTS: One hundred ppm of NO was statistically equivalent to the heparin-coated circuit for attenuating bypass-induced blood activation, and a combination of the two significantly surpassed the results of either modification alone. Fifty ppm of NO alone provided only a slight attenuation of blood activation as compared with the non-heparin-coated circuit, though the difference was not significant. A combination of 50 ppm NO and the heparin-coated circuit did not significantly enhance the effects of the heparin-coated circuit alone. CONCLUSIONS: The combination of NO gas infusion and heparin-coated circuits appears to be a useful and promising modification for enhancing the attenuation of bypass-induced blood activation, though the optimal dose of NO infusion in terms of effectiveness and adverse effects to the whole body remains to be established.

Complement activation in circulation and central nervous system after rituximab (anti-CD20) treatment of B-cell lymphoma.

Rituximab (IDEC-C2B8, Mabthera, Rituxan), a chimeric monoclonal antibody against the B-cell specific CD20-antigen, has been demonstrated to be effective in the treatment of non-Hodgkin's B-cell lymphoma (B-NHL). Previous in vitro studies have shown that direct complement-dependent cytotoxicity (CDC), ADCC and apoptosis are important in the rituximab-induced killing of lymphoma cells. It is, however, unknown whether rituximab penetrates the blood-brain barrier. Therefore, we studied rituximab levels and complement (C) activation in blood and cerebrospinal fluid (CSF) following intravenous rituximab therapy in a patient with relapsing non-Hodgkin's lymphoma with central nervous system (CNS) involvement. Longitudinal samples from blood and CSF were taken at 13 time-points during the treatment period. The results show that the C cascade becomes activated in blood during the first mAb infusion (C3a-desArg concentration rose from 55 to 138 microg/ml during the first 2 hours). After the first infusion the proportions of lymphocytes positive for the CD19- and CD20-antigens in the peripheral blood were reduced from 41% and 35%, respectively, to a level of 2% (for both). In CSF the rituximab concentration increased after successive infusions, but remained below 0.55 microg/ml (compared to a Cmax of 400 microg/ml in peripheral blood). Although a minor and delayed C activation response was seen in the CSF the treatment did not clear CD20-positive cells away from the CNS. Thus, it appears that an intact blood-brain barrier restricts the entry of rituximab into the CNS. Possible options to circumvent this would be dose escalation or intrathecal rituximab treatment.

Biocompatibility of hemodialysis membranes: interrelations between plasma complement and cytokine levels.

Hemodialysis (HD) membrane biocompatibility is defined as absence of complement activation. We have recently shown that circulating levels of interleukin (IL) 1 and IL-2 predict death and survival, respectively, of HD patients. Studies have assessed IL-1 in treatments with biocompatible and less biocompatible dialysis membranes, but no study has correlated circulating levels of all these immunoreactants. We assessed these immunoreactants, and temperature as an outcome, during HD in patients treated with different membranes. Twelve stable patients, receiving thrice-weekly chronic bicarbonate HD, were randomly dialyzed with three different types of membranes, composed of: Cuprophan, cuprammonium rayon modified cellulose, and Hemophan. Blood was drawn from the arterial line port before (Pre) and 15, 30, and 60 min during and after (Post) HD. Patients' temperatures were measured before and after each treatment. The plasma concentrations of IL-1 and IL-2 and factors C3a and C5a were assessed by ELISA. There were no differences between baseline levels of any of the immunoreactants in patients treated with different dialyzers. C3a, C5a, and IL-1 levels increased significantly during HD treatments with all three different membranes. C3a, C5a, and IL-1 levels during Cuprophan and Hemophan treatments were significantly higher than the levels during modified cellulose treatment at 30 and 60 min and Post (p < 0.01). For all the immunoreactants, however, the Post levels were higher than the Pre levels. In contrast to IL-1, there were no differences in mean IL-2 levels during treatments when different membranes were compared. There were few correlations of plasma C3a and C5a levels with plasma IL-1 levels, but there was only one treatment time in one dialyzer group during which IL-2 and any of the other factors were correlated. Pre and Post temperature values and percent change in temperature were not correlated with any of the immunoreactants measured. These data show that C3a, C5a, and IL-1 responses are similar, but not identical, during treatments with different membranes. The response of circulating IL-2 levels to treatments is quite different from that of plasma C3a, C5a and IL-1 levels and suggests that these changes are not solely due to treatment factors. Treatment with modified cellulose membranes is associated with a different immunoreactive profile as compared with patients dialyzed using other cellulose membranes. We suggest that circulating IL-1 levels are good biocompatibility markers.

Tumor necrosis factor alpha release is a major biological event associated with rituximab treatment.

INTRODUCTION: In patients with low-grade non-Hodgkin's lymphoma, rituximab (MabThera) produces infusion-related toxicity, including fever, rigors, and chills in greater than 50% of those treated. The majority of these reactions are grade 1 or 2. MATERIALS AND METHODS: In the GELA study LNH98-5, a total of 400 elderly patients with previously untreated diffuse large B-cell lymphoma were randomized to treatment with CHOP or with rituximab plus CHOP (R-CHOP). In a detailed investigation of biological events which may be associated with adverse reactions specific to rituximab infusion, a subgroup of 55 patients (26 in the CHOP group and 29 in the R-CHOP group) were selected for measurement of several biological parameters at baseline and at 1, 4 and 8 h (H1, H4 and H8, respectively) after commencing therapy. For 27 patients, measurements included cytokine and complement levels. RESULTS: Baseline demographic and disease characteristics were similar for patients in both treatment groups. Compared with the CHOP treatment group, patients in the R-CHOP group had significantly higher post-treatment changes in neutrophil, lymphocyte, and monocyte counts, LDH levels, C3a levels, and TNF-alpha levels. In the R-CHOP group, neutrophil levels increased at H4 (P<0.05), lymphocyte levels decreased at H1 (P<0.05), H4 (P<0.001) and H8 (P<0.05), monocytes levels decreased at H1 (P<0.01), LDH levels increased at H4 (P<0.05) and H8 (P<0.01), and C3a decreased at H1 (P<0.01). The most statistically significant changes were observed for TNF-alpha levels: Mean values of TNF-alpha increased more than 250% at H1 and H4 and were still increased by 170% at H8 (P<0.001 at all timepoints). Since only six of the 55 evaluated patients had severe adverse events, it was not possible to correlate severe toxicity with these biological variations. CONCLUSION: This analysis demonstrates that rituximab infusion was rapidly followed by activation of complement, B-lymphocyte cytolysis, and TNF-alpha release.

Selective blockade of membrane attack complex formation during simulated extracorporeal circulation inhibits platelet but not leukocyte activation.

OBJECTIVE: Complement activation is induced by cardiopulmonary bypass, and previous work found that late complement components (C5a, C5b-9) contribute to neutrophil and platelet activation during bypass. In the present study, we blocked C5b-9 formation during extracorporeal recirculation of whole blood to assess whether the membrane attack complex was responsible for both platelet and leukocyte activation. METHODS: In a simulated extracorporeal model that activates complement (C3a and sC5b-9), platelets (CD62P expression, leukocyte-platelet conjugate formation), and leukocytes (increased CD11b expression and neutrophil elastase), we examined an anti-human C8 monoclonal antibody that inhibits C5b-9 generation for its effects on cellular activation. RESULTS: Anti-C8 significantly inhibited sC5b-9 formation but did not block C3a generation. Anti-C8 also significantly inhibited the increase in platelet CD62P and monocyte-platelet conjugate formation seen with control circulation. Moreover, compared with control circulation, in which the number of circulating platelets fell by 45%, addition of anti-C8 completely preserved platelet counts. In contrast to blockade of both C5a and sC5b-9 during simulated extracorporeal circulation, neutrophil activation was not inhibited by anti-C8. However, circulating neutrophil and monocyte counts were preserved by addition of anti-C8 to the extracorporeal circuit. CONCLUSIONS: The membrane attack complex, C5b-9, is the major complement determinant of platelet activation during extracorporeal circulation, whereas C5b-9 blockade has little effect on neutrophil activation. These data also suggest a role for platelet activation or C5b-9 (or both) in the loss of monocytes and neutrophils to the extracorporeal circuit.

Suppression of alternative complement pathway activity by radiographic contrast media.

The authors examined the effect of four different kinds of contrast media (ionic/non-ionic, monomer/dimer) on the activation of the complement (C) system (haemolytic activity and anaphylatoxin generation) in vitro. In addition, the authors compared the effect of contrast media on inulin-mediated generation of the anaphylatoxin derivative C3a des Arg in sera from urticarial reactors and their non-reacting controls. It was observed that the incubation of commercial iohexol, ioxaglate, iodixanol and meglumin amidotriz solutions in normal human serum (NHS) resulted in a dose-dependent decrease in the haemolytic activity of the alternative C pathway. Contrary to expectations the contrast media did not activate C in NHS. Instead, inulin-induced generation of C3a des Arg was inhibited by all the four contrast media. The strongest inhibitor was ioxaglate, an ionic dimer. No significant difference between the urticarial reactors and non-reactors in the inhibition of C3a des Arg generation was observed. In analyzing the mechanism of C inhibition we found that the contrast media solutions, particularly the ionic ones, prevented formation of the alternative pathway C3 convertase, C3bBb, by inhibiting the binding of factor B to surface-associated C3b molecules. The results suggest that the previously observed decrease in haemolytic C titres by contrast media is due to direct suppression of C activity rather than activation-induced consumption.

Effects of high-dose intraperitoneal aprotinin treatment on complement activation and acute phase response in acute severe pancreatitis.

Forty-eight patients with severe acute pancreatitis were treated with intraperitoneal lavage in a double-blind randomized multi-center trial. One group (aprotinin group, n = 22) was also treated intraperitoneally with high doses of the protease inhibitor aprotinin. In the group not treated with aprotinin (control group), 6 patients were operated on because of pancreatic necrosis, compared with none in the treated group. Complement activation and the acute phase response were studied with measurements of anaphylatoxin C3a, C1 inhibitor (C1 Inh), interleukin 6 (IL-6), and C-reactive protein (CRP). The control group had higher plasma levels of C3a and lower levels of C1 Inh compared with the aprotinin group. The differences were statistically significant for C3a but not for C1 Inh. Both groups had high plasma levels of IL-6 and CRP. There were no differences between the groups in CRP levels, but the control group had higher IL-6 levels (not statistically significant) than the aprotinin group. This was caused by very high levels in the 6 patients operated on because of pancreatic necrosis, indicating that IL-6 could be a good plasma marker of pancreatic necrosis. The results also show that massive antiprotease treatment reduces complement activation, as illustrated by the lower C3a levels in the aprotinin group. The lower C1 Inh levels in the control group could have been caused by an increased consumption of the inhibitor.

Heparin-coated cardiopulmonary bypass circuits in coronary bypass surgery.

Cardiopulmonary bypass (CPB) is a nonphysiologic environment for an organism. The damage of blood components may also lead to organ dysfunction, some-times recognized as postperfusion syndrome. One possible way to diminish the risk of these complications would be to reduce the thorombogenicity and to improve the biocompatibility of the artificial surfaces by using a heparin-coated CPB circuit. In this study, we compared a heparin-coated CPB circuit with a noncoated CPB circuit in terms of biocompatibility in 20 patients undergoing elective coronary bypass surgery. We employed a Duraflo II (n = 10) as a heparin-coated CPB circuit and a Univox IC (n = 10) as control subjects. Ten patients (Group C) were operated on using the heparin-coated CPB circuit. A total of 10 patients were given heparin in a reduced dose (2.0 mg/kg), and additional heparin was given if the activated clotting time (ACT) was below 400 s. The control group also included 10 patients (Group NC), who were operated on with noncoated devices. They received 2.5 mg/kg of heparin, and additional heparin was given if the ACT was below 450 s. All patients had normal coagulation parameters and did not receive blood transfusion. We measured complement activation levels (C3a, C4a), platelet count, thrombin-antithrombin III complex levels, D-dimer levels, and ACT during CPB and respiratory index postoperatively. The concentration of C3a in group NC was significantly higher than that in group C. Platelet reduction in group NC was significantly greater than that in group C. There were no significant differences in the remaining parameters between the 2 groups. We concluded that heparin-coated CPB circuits improved biocompatibility by reducing complement activation and platelet consumption and enabled us to reduce the dose of heparin required for systemic heparinization.

Alkylation of cellulosic membranes results in reduced complement activation.

4-Vinyl pyridine was grafted to the surface of the cellulosic membrane Cuprophan, and subsequently alkylated with both C10 and C16 aliphatic chains. Complement activation of heparinized human blood, corrected for anaphylatoxin adhesion, was measured by radioimmunoassay. The surface treatments both yielded substantial reductions in C5a activity, with a lessor reduction in C3a and C4a activity. Alkylation with 10 and 16 carbon chains resulted both in enhancements of albumin adsorption and stability. These enhancements as well as the reductions in complement activation were statistically indistinguishable between the two treatments. The reduction in complement activation was influenced more by adsorption of endogenous albumin and possibly by the vinyl pyridine graft, than the removal of surface active hydroxyl groups from Cuprophan.

Influence of steroids on complement and cytokine generation after cardiopulmonary bypass.

BACKGROUND: It is recognized that the inflammatory mediators complement and cytokines are generated during cardiopulmonary bypass as an endogenous response to extracorporeal circulation. METHODS: Nineteen randomized patients (10 steroid/9 nonsteroid) entered an institutional review board-approved protocol to measure complement and interleukin level generation before and after elective coronary revascularization. The steroid regimen involved 1 g of methylprednisolone sodium succinate intravenously before bypass and 4 mg of dexamethasone every 6 hours for four doses during the first 24 hours of recovery. Complement and interleukin levels were measured before bypass, immediately after bypass, and at 24, 48 and 72 hours of recovery. RESULTS: In the nonsteroid group, there was a significant elevation in all inflammatory mediators relative to the steroid group. The predominant changes occurred at 24 hours after operation. CONCLUSIONS: Steroids produced a dramatic reduction in complement and interleukin levels. The number of patients was clearly too small to document a clinical consequence of steroid administration.

Experimental studies of the influence of priming solutions on the systemic activation of complement during cardiopulmonary bypass: comparison between the use of albumin, hydroxyethyl starch and HWA-138.

Cardiopulmonary bypass surgery may be complicated by a systemic inflammatory reaction, which has been ascribed to activation of complement. For such activation, the choice of priming solution for the heart-lung machine may be of importance. The peripheral blood of three groups of eight donors was exposed to albumin, hydroxyethyl starch (HES) or to HWA-138 (pentoxifylline analogue) in addition to the priming solutions. The study confirmed that activation of complement is a consistent phenomenon during cardiopulmonary bypass surgery. The concentration of the C3 activation product C3a in the plasma was significantly increased after simulated extracorporeal circulation. However there were no differences within the increase of C3a concentrations between the various priming solutions.