Ocular and Systemic Complement Activation during Anti-Vascular Endothelial Growth Factor Treatment and Age-Related Eye Disease Study 2 Dietary Supplementation in Neovascular Age-Related Macular Degeneration.

PURPOSE: The aim of this study was to investigate the influence of dietary supplementation using Age-Related Eye Disease Study 2 (AREDS2) on complement activation in patients with neovascular age-related macular degeneration (nAMD) under ongoing treatment. METHODS: In this prospective, single-center, controlled, open-label investigator-initiated trial, eligible nAMD patients were randomized at a ratio of 1:1 in 2 groups: those with and without dietary AREDS2 supplementation for 4 weeks. Zinc, plasma, and aqueous humor (AH) complement levels were quantified via enzyme-linked immunosorbent assays. RESULTS: Fifty of 62 enrolled patients completed the trial (AREDS2 n = 27, controls n = 23). Systemic zinc and complement levels were not different at baseline between the 2 groups (p > 0.1). At the final visit, systemic zinc levels were significantly higher in the AREDS2 group (10.16 ± 2.08 µmol/L; 8.66 ± 1.17 µmol/L; p = 0.007), whereas systemic and AH complement levels were not different (p > 0.1). In both groups, no significant change was observed in systemic levels of C3, C3a, FH, FI, and sC5b-9 (p > 0.1). Only systemic complement component Ba showed an increase from baseline to the end visit (p = 0.01). This increase was higher in the control group (p = 0.02) than in the AREDS2 group (p = 0.23). CONCLUSIONS: Short-term dietary AREDS2 supplementation leads to a significant increase in systemic zinc levels without any influence on complement activation levels.

Structure of a homofructosan from Saussurea costus and anti-complementary activity of its sulfated derivatives.

A homogeneous water-soluble polysaccharide APS-W1, (2→1)-ß-d-fructofuranosan, with an average molecular weight of 3.9kDa, was isolated and characterized from the roots of Saussurea costus. Five sulfated derivatives of APS-W1 with different degrees of sulfation were prepared and they showed strong inhibitory effect on the complement activation through the classical pathway (CP50: 2.2-18.9µg/mL; 8.3µg/mL for heparin) and alternative pathway (AP50: 11.4-115.8µg/mL; 89.2µg/mL for heparin). Mechanism studies by using complement-depleted sera indicated that sulfated derivatives with different positions of sulfation targeted to different complement proteins. Meanwhile the sulfated derivatives have limited anticoagulant effect based on re-calcification time and thrombin time. These results suggested that the sulfated derivatives prepared from APS-W1 could be promising potential complement inhibitors for the treatment of diseases caused by an over-activated complement system.

Rapid activation of monocyte tissue factor by antithymocyte globulin is dependent on complement and protein disulfide isomerase.

Lymphocyte depletion with antithymocyte globulin (ATG) can be complicated by systemic coagulation activation. We found that ATG activated tissue factor procoagulant activity (TF PCA) on monocytic cells more potently than other stimuli that decrypt TF, including cell disruption, TF pathway inhibitor inhibition, and calcium ionophore treatment. Induction of TF PCA by ATG was dependent on lipid raft integrity and complement activation. We showed that ATG-mediated TF activation required complement activation until assembly of the C5b-7 membrane insertion complex, but not lytic pore formation by the membrane attack complex C5b-9. Consistently, induction of TF PCA by ATG did not require maximal phosphatidylserine membrane exposure and was not correlated with the magnitude of complement-induced lytic cell injury. Blockade of free thiols, an inhibitory monoclonal antibody to protein disulfide isomerase (PDI), and the small-molecule PDI antagonist quercetin-3-rutinoside prevented ATG-mediated TF activation, and C5 complement activation resulted in oxidation of cell surface PDI. This rapid and potent mechanism of cellular TF activation represents a novel connection between the complement system and cell surface PDI-mediated thiol-disulfide exchange. Delineation of this clinically relevant mechanism of activation of the extrinsic coagulation pathway during immunosuppressive therapy with ATG may have broader implications for vascular thrombosis associated with inflammatory disorders.

Role of complement in a murine model of peanut-induced anaphylaxis.

Peanut allergy is severe and persisting from childhood to adulthood. However, there is no effective prophylaxis or treatment for peanut allergy. Little is known to about the molecular process in the pathogenesis of peanuts allergy, especially in innate immunity. Thus we investigated the role of complement activation in murine peanut anaphylaxis. Complement component C3 deposition on peanut extract (PE) was evaluated using sera from wild-type (WT), mannose-binding lectin associated serine protease (MASP)-1/3 deficient, MASP-2 deficient, and C4 deficient mice. Sera from interferon regulatory factor-4 (IRF-4) deficient mice, which lack serum immunoglobulin, were also used. In anaphylaxis study, mice were pretreated with propranolol and a long-acting form of IL-4, and injected with PE. Mice were then assessed for plasma C3a levels and hypothermia shock by ELISA and rectal temperature measurement, respectively. C3 deposition on PE was abolished in immunoglobulin- and C4-deficient sera. No difference in C3 deposition levels were observed among WT, MASP-1/3 deficient and MASP-2 deficient sera. IgM, IgG2b, IgG3, C1q, and ficolin-A deposits were detected on PE. In anaphylaxis study, MASP-1/3 deficient mice showed elevation of plasma C3a levels similar to WT mice. However, they were significantly reduced in C4- and MASP-2-deficient mice compared to WT mice. Consistently, PE-induced anaphylactic shock was prevented in C4 deficient mice and partially in MASP-2 deficient mice. In conclusion, PE activates complement via both the lectin and classical pathways in vivo, and the complement activation contributes to hypothermia shock in mice.

Complement activation on the surface of cell-derived microparticles during cardiac surgery with cardiopulmonary bypass - is retransfusion of pericardial blood harmful?

OBJECTIVES: To investigate whether cell-derived microparticles play a role in complement activation in pericardial blood of patients undergoing cardiac surgery with cardiopulmonary bypass (CPB) and whether microparticles in pericardial blood contribute to systemic complement activation upon retransfusion. METHODS: Pericardial blood of 13 patients was retransfused in 9 and discarded in 4 cases. Microparticles were isolated from systemic blood collected before anesthesia (T1) and at the end of CPB (T2), and from pericardial blood. The microparticles were analyzed by flow cytometry for bound complement components C1q, C4 and C3, and bound complement activator molecules C-reactive protein (CRP), serum amyloid P-component (SAP), immunoglobulin (Ig)M and IgG. Fluid-phase complement activation products (C4b/c, C3b/c) and activator molecules were determined by ELISA. RESULTS: Compared with systemic T1 blood, pericardial blood contained increased C4b/c and C3b/c, and increased levels of microparticles with bound complement components. In systemic T1 samples, microparticle-bound CRP, whereas in pericardial blood, microparticle-bound SAP and IgM were associated with complement activation. At the end of CPB, increased C3b/c (but not C4b/c) was present in systemic T2 blood compared with T1, while concentrations of microparticles binding complement components and of those binding complement activator molecules were similar. Concentrations of fluid-phase complement activation products and microparticles were similar in patients whether or not retransfused with pericardial blood. CONCLUSIONS: In pericardial blood of patients undergoing cardiac surgery with CPB, microparticles contribute to activation of the complement system via bound SAP and IgM. Retransfusion of pericardial blood, however, does not contribute to systemic complement activation.

The complement cascade: Yin-Yang in neuroinflammation--neuro-protection and -degeneration.

The complement cascade has long been recognized to play a key role in inflammatory and degenerative diseases. It is a 'double edged' sword as it is necessary to maintain health, yet can have adverse effects when unregulated, often exacerbating disease. The contrasting effects of complement, depending on whether in a setting of health or disease, is the price paid to achieve flexibility in scope and degree of a protective response for the host from infection and injury. Loss or even decreased efficiency of critical regulatory control mechanisms can result in aggravated inflammation and destruction of self-tissue. The role of the complement cascade is poorly understood in the nervous system and neurological disorders. Novel studies have demonstrated that the expression of complement proteins in brain varies in different cell types and the effects of complement activation in various disease settings appear to differ. Understanding the functioning of this cascade is essential, as it has therapeutic implications. In this review, we will attempt to provide insight into how this complex cascade functions and to identify potential strategic targets for therapeutic intervention in chronic diseases as well as acute injury in the CNS.

Immune stimulating properties of a novel polysaccharide from the medicinal plant Tinospora cordifolia.

An alpha-D-glucan (RR1) composed of (1-->4) linked back bone and (1-->6) linked branches with a molecular mass of >550 kDa and exhibiting unique immune stimulating properties is isolated and characterized from the medicinal plant Tinospora cordifolia. This novel polysaccharide is noncytotoxic and nonproliferating to normal lymphocytes as well as tumor cell lines at 0-1000 microg/ml. It activated different subsets of the lymphocytes such as natural killer (NK) cells (331%), T cells (102%), and B cells (39%) at 100 microg/ml concentration. The significant activation of NK cells is associated with the dose-dependent killing of tumor cells by activated normal lymphocytes in a functional assay. Immune activation by RR1 in normal lymphocytes elicited the synthesis of interleukin (IL)-1beta (1080 pg/ml), IL-6 (21,833 pg/ml), IL-12 p70 (50.19 pg/ml), IL-12 p40 (918.23 pg/ml), IL-18 (27.47 pg/ml), IFN- gamma (90.16 pg/ml), tumor necrosis factor (TNF)-alpha (2225 pg/ml) and monocyte chemoattractant protein (MCP)-1 (2307 pg/ml) at 100 microg/ml concentration, while it did not induce the production of IL-2, IL-4, IL-10, interferon (IFN)-alpha and TNF-beta. The cytokine profile clearly demonstrates the Th1 pathway of T helper cell differentiation essential for cell mediated immunity, with a self-regulatory mechanism for the control of its overproduction. RR1 also activated the complements in the alternate pathway, demonstrated by a stepwise increase in C3a des Arg components. Incidentally, RR1 stimulation did not produce any oxidative stress or inducible nitric oxide synthase (iNOS) in the lymphocytes or any significant increase in nitric oxide production. The water solubility, high molecular mass, activation of lymphocytes especially NK cells, complement activation, Th1 pathway-associated cytokine profile, together with a low level of nitric oxide synthesis and absence of oxidative stress confer important immunoprotective potential to this novel alpha-D-glucan.

Neutrophils, not complement, mediate the mortality of experimental hemorrhagic pancreatitis.

Chemoactivation of the neutrophil (PMN) via the complement system has been observed in many inflammatory conditions and is thought to play a pathogenic role in acute pancreatitis. This study examined the effects of PMN depletion in experimental hemorrhagic pancreatitis and tested the role played by complement. Severe pancreatitis was induced by a choline-deficient, 0.5% ethionine-supplemented diet in female Institute of Cancer Research (ICR) mice weighing 11-13 g. Neutropenia was induced by an antibody injection. Total complement depletion was achieved by tail vein injections of cobra venom factor (CVF). Serum amylase levels and local pancreatic injury were not significantly modulated by either PMN or complement depletion at 72 hours. Systemic and remote organ injury, assessed by the formation of ascites, hematocrit, and serum alanine aminotransferase levels, was significantly reduced in neutropenic mice but failed to be moderated by complement depletion. In addition, liver and lung myeloperoxidase activity was independent of complement depletion. At 5 days, mortality was zero in PMN-depleted mice. There was no improvement in survival in the CVF-treated group. Neutrophils are important in the systemic injury and mortality of severe pancreatitis. PMN chemoactivation involves mechanisms other than complement.

[Modulation of the inflammatory response through complement-neutrophil activation feedback mechanism with selenium and vitamin E].

To identify the regulatory effect of sodium selenite and vitamin E on the complement-neutrophil-reactive oxygen(ROS) activation feedback (CNAF) mechanism mediated inflammatory response, we detected ROS production and complement activation in vitro tests by chemiluminescence technique and complement fixation and recognized the regulation of the inflammatory response in vivo mouse vasculitis models of skin, lung, and liver. Convincing results were observed as both in vitro and in vivo experiments showing inhibition of CNAF mechanism with sodium selenite and vitamin E could effect the reduced ROS production and complement activation. The incidence (100%) for vasculitis in control group decreased to 20%-57% in sodium selenite and vitamin E treated groups. Elucidation of the ancillary mechanism of CNAF enhancing inflammatory response is a promising area for new therapeutic developments in the modulation of inflammatory response. As in a clinical approach, a remarkable therapeutic effect with sodium selenite was observed during an epidemic episode of epidemic hemorrhagic fever in Henan province. The mortality rate of fulminant cases was reduced from 100% of untreated control cases to 36.6% by treatment with sodium selenite. The results of present studies strongly suggest that antioxidants such as selenium and vitamin E as well as others like flavonoids can exhibit a novel anti-inflammatory action via this CNAF mechanism. It is expected in the future an increasing number of patients with severe infections or inflammatory disorders in which excessive complement activation and adverse ROS production have been implicated, e. g. ischemia-reperfusion injury, severe sepsis and diverse inflammatory vascular injuries like rheumatoid arthritis, hepatitis and inflammatory bowel diseases should benefit from this newer concept guided adjuvant therapies which make use of nutrient antioxidants like selenium, vitamin E and others.

Changes in immunomodulatory activity of human mononuclear cells after cultivation with leaf decoctions from the genus Ligustrum L.

Leaf decoctions from Ligustrum vulgare (LV) and Ligustrum delavayanum (LD) were studied for candidacidal activity, phagocytic activity studied on human mononuclear cells (MO) and complement activated by the classical pathway. Candidacidal activity was studied on Candida albicans SC 1539 incubated with MO. The decoction of LD increased the candidacidal activity of MO, whereas LV did not show any effect. The phagocytic activity of MO was decreased by the decoction of LV, whereas LD did not change the activity. The phagocytic index of MO incubated with LD decoction was increased, but use of the LV decoction did not show significant changes. Decoctions from LD and LV significantly decreased the haemolytic activity of complement activated by the classical pathway (conc. 0.78 mg/mL).

[The inhibitory effect of selenite and other antioxidants on the complement mediated experimental pulmonary alveolitis].

OBJECTIVE: To test the regulatory effect of selenite and other antioxidants on complement PMN activation feedback mechanism mediated inflammatory response in experimantal pulmonary vasculitis. METHODS: Mouse model of pulmonary Arthus reaction vasculitis developed with BSA sensitization and antigen inhalation was used for tests of inhibitory effect of oral administration of sodium selenite, glycyrrhizin flavonoids and/or vitamin E on the incidence of developing vasculitis, which could reflect the modulation of inflammatory response and tissue injury. RESULTS: A remarkable inhibitory effect of selenite or combined flavonoid and vitamin E on the induction of pulmonary vasculitis were observed as the incidence of vasculitis could drop from 100% of the untreated group down to 25% of the antioxidants treated groups, revealing modulation of inflammatory response. CONCLUSIONS: It indicated a newer insight into mechanism of the inflammatory response could give a novel approach to the modulation of inflammatory response. An adjuvant therapy of selenium and antioxidants to the interventions of inflammatory disorders has been expected.

A blockade of complement activation prevents rapid intestinal ischaemia-reperfusion injury by modulating mucosal mast cell degranulation in rats.

We attempted to define the putative role of complement activation in association with mucosal mast cell (MMC) degranulation in the pathogenesis of rapid intestinal ischaemia-reperfusion (I/R) injury. We prepared complement activity-depleted rats by the administration of the anti-complement agent K-76COOH and the serine-protease inhibitor FUT-175. Autoperfused segments of the jejunum were exposed to 60 min of ischaemia, followed by reperfusion for various time periods, and the epithelial permeability was assessed by the 51Cr-EDTA clearance rate. The number of MMC was immunohistochemically assessed. In control rats, the maximal increase in mucosal permeability was achieved by 30-45 min of reperfusion. This increase was significantly attenuated by the administration of either K-76COONa alone or in combination with FUT-175. In contrast, the administration of carboxypeptidase inhibitor (CPI), which prevents the inactivation of complement-derived anaphylatoxins such as C5a, significantly enhanced the increase in I/R-induced mucosal permeability. These findings were confirmed morphologically by light microscopy and scanning electron microscopy. In addition, the I/R-induced mucosal injury was accompanied by a marked decrease in the number of MMC, and administration of K-76COOH significantly inhibited this change. These results indicate that complement activation and the generation of complement-derived anaphylatoxins are key events in I/R-induced mucosal injury. It is likely that intestinal I/R-induced mucosal injury may be partially mediated by MMC activation associated with the complement activation.

Cora rotary pump for implantable left ventricular assist device: biomaterial aspects.

Our group is developing a left ventricular assist device based on the principle of the Maillard-Wankel rotative compressor: it is a rotary, not centrifugal, pump that produces a pulsatile flow. Stringent requirements have been defined for construction materials. They must be light, yet sufficiently hard and rigid, and able to be machined with high precision. The friction coefficient must be low and the wear resistance high. The materials must be chemically inert and not deformable. Also, the materials must be biocompatible, and the blood contacting surface must be hemocompatible. We assessed the materials in terms of physiochemistry, mechanics, and tribology to select the best for hemocompatibility (determined by studies of protein adsorption; platelet, leukocyte, and red cell retention; and hemolysis, among other measurements) and biocompatibility (determined by measurement of complement activation and toxicity, among other criteria). Of the materials tested, for short- and middle-term assistance, we chose titanium alloy (Ti6Al4V) and alumina ceramic (Al2O3) and for long-term and permanent use, composite materials (TiN coating on graphite). We saw that the polishing process of the substrate must be improved. For the future, the best coating material would be diamond-like carbon (DLC) or crystalline diamond coating.

Stabilization of rat heart mitochondria by alpha-tocopherol in rats.

alpha-Tocopherol (alpha-T) acts by different mechanisms: scavenging free oxygen radicals (FOR) and stabilizing membranes; both may contribute to the prevention of the pathogenesis of diseases like cardiovascular events. The present work shows that alpha-T breaks the vicious cycle generated by damaged mitochondria. The latter stimulate the immune system increasing FOR formation that leads to more damaged mitochondria. alpha-T inhibits lipid peroxidation induced by recombinant human tumor necrosis factor (rh-TNF). Polymorphonuclear neutrophils (PMN) are activated by TNF to produce FOR causing lipid peroxidation of cardiomyocyte membranes. Since TNF is used as a therapeutic agent against certain tumors, treating patients with alpha-T could protect them against systemic side effects of TNF.

[Comparative analysis of various plasmapheresis methods--modern procedures of mechanical plasma collection compared with each other and with manual bag centrifugation procedures]. / Vergleichsanalyse verschiedener Plasmapheresemethoden--Moderne Verfahren apparativer Plasmagewinnung im Vergleich untereinander und mit dem manuellen Beutel-Zentrifugationsverfahren.

Four plasmapheresis procedures (manual blood bag centrifugation plasmapheresis, and the three plasmapheresis machines P.C.S./Haemonetics, Autopheresis-C/Baxter-Travenol, Plasmapur Monitor/Organon Teknika) were studied comparatively. The three machine procedures could be performed more easily and more rapidly and were well accepted by donors, autologous donors (patients) and staff. Compared with the traditional, well established manual procedure, a possible impairment of the plasma donors seems reduced rather than raised. Activation of the hemostatic system of the donors, measured with very sensitive methods, was found to be less pronounced when plasmapheresis was performed with the machines than when it was performed conventionally. The plasma product obtained by machine plasmapheresis was found to be of higher quality. All three systems showed less activation of the clotting system. Especially the plasma obtained by the P.C.S. showed a higher clotting factor yield. Plasma obtained by the Autopheresis-C and by the Plasmapur Monitor (both systems are equipped with filters) was markedly less contaminated with cells. The recently found low activation of the clotting system of plasma recipients, however, showed no differences when plasma obtained conventionally or by the Plasmapur Monitor was used. In summary, the new machine plasmapheresis devices offer a good alternative to the conventional blood bag centrifugation method and set new standards for the production of high quality plasma.

[Mediators and ARDS]. / Mediatoren und ARDS.

The symptoms of adult respiratory distress syndrome (ARDS) include dyspnea, tachypnea, hypoxemia refractory to supplemental oxygen and bilateral infiltrations in the chest X-ray. Neutrophils are implicated in the pathogenesis as important effector cells acting by release of mediators. Activation of the complement system has been shown in several studies and can induce lung damage directly in animal models. Proteases and collagenase have been found in elevated concentration in bronchoalveolar lavage fluid, while the amount of protease-inhibitors has been found to be reduced. Arachidonic acid metabolites of the cyclooxygenase and lipoxygenase pathway, such as prostaglandins and leukotrienes, may play a role in the pathogenesis or perpetuation of the disease process. The same holds true for cytokines such as interleukin-1 or tumor necrosis factor. All of them have been found to be elevated either in plasma or bronchoalveolar lavage fluid of ARDS patients. Several lines of evidence implicate oxygen radicals as important mediators of lung damage in ARDS. The therapeutic implications of these new insights into the pathogenesis of ARDS are briefly discussed.

Complement activation and reinfusion of wound drainage blood.

Eighteen patients undergoing total hip replacement (n = 13) or knee arthroplasty (n = 5) due to osteoarthritis or osteoarthrosis were prospectively studied in an investigation of complement activation and anaphylatoxin release in association with reinfusion of aspirated wound blood. Twelve of the patients needed blood transfusions and received an average of 390 +/- 75 ml (+/- SD) of autologous blood within 45 min. Plasma complement components, anaphylatoxins, and inhibitors were studied 1 min before and 15 min after the start of and 15 min after the completion of autologous transfusion. Samples also were taken from the collected blood, before and after passing it through a microporous filter. Blood gases and systemic complement samples were drawn simultaneously. There were no significant changes in systemic complement variables before, during, or after transfusion of autologous blood. However, in the aspirated blood, increased concentration of anaphylatoxins (C3a and C5a) and terminal complement complexes (TCC) were present (P less than 0.001). There were no differences observed between samples drawn before and after filtration of the blood. The concentration of C5 was less in the collected blood than in the systemic blood (P less than 0.05). No changes in blood gases were observed. This study demonstrated that postoperatively salvaged whole blood underwent anaphylatoxin formation and complement activation. However, after reinfusion of this blood, neither systemic complement activation nor clinical complications were observed.