Down-regulation of complement receptors on the surface of host monocyte even as in vitro complement pathway blocking interferes in dengue infection.

In dengue virus (DENV) infection, complement system (CS) activation appears to have protective and pathogenic effects. In severe dengue fever (DF), the levels of DENV non-structural-1 protein and of the products of complement activation, including C3a, C5a and SC5b-9, are higher before vascular leakage occurs, supporting the hypothesis that complement activation contributes to unfavourable outcomes. The clinical manifestations of DF range from asymptomatic to severe and even fatal. Here, we aimed to characterise CS by their receptors or activation product, in vivo in DF patients and in vitro by DENV-2 stimulation on monocytes. In comparison with healthy controls, DF patients showed lower expression of CR3 (CD11b), CR4 (CD11c) and, CD59 on monocytes. The DF patients who were high producers of SC5b-9 were also those that showed more pronounced bleeding or vascular leakage. Those findings encouraged us to investigate the role of CS in vitro, using monocytes isolated from healthy subjects. Prior blocking with CR3 alone (CD11b) or CR3 (CD11b/CD18) reduced viral infection, as quantified by the levels of intracellular viral antigen expression and soluble DENV non-structural viral protein. However, we found that CR3 alone (CD11b) or CR3 (CD11b/CD18) blocking did not influence major histocompatibility complex presentation neither active caspase-1 on monocytes, thus probably ruling out inflammasome-related mechanisms. Although it did impair the secretion of tumour necrosis factor alpha and interferon alpha. Our data provide strategies of blocking CR3 (CD11b) pathways could have implications for the treatment of viral infection by antiviral-related mechanisms.

AAV-mediated expression of human PRELP inhibits complement activation, choroidal neovascularization and deposition of membrane attack complex in mice.

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Approximately 50% of AMD patients have a polymorphism in the negative regulator of complement known as Factor H. Individuals homozygous for a Y402H polymorphism in Factor H have elevated levels of membrane attack complex (MAC) in their choroid and retinal pigment epithelium relative to individuals homozygous for the wild-type allele. An inability to form MAC due to a polymorphism in C9 is protective against the formation of choroidal neovascularization (CNV) in AMD patients. Hence, blocking MAC in AMD patients may be protective against CNV. Here we investigate the potential of human proline/arginine-rich end leucine-rich repeat protein (PRELP) as an inhibitor of complement-mediated damage when delivered via the subretinal route using an AAV2/8 vector. In a fluorescence-activated cell sorting (FACS) lysis assay, PRELP inhibited normal human serum-mediated lysis of Hepa-1c1c7 cells by 18.7%. Unexpectedly, PRELP enhanced the formation of tubes by human umbilical vein endothelial cells (HUVECs) by approximately 240%, but, when delivered via an AAV vector to the retina of mice, PRELP inhibited laser-induced CNV by 60%. PRELP reduced deposition of MAC in vivo by 25.5%. Our results have implications for the development of complement inhibitors as a therapy for AMD.

Aluminum hydroxide adjuvant differentially activates the three complement pathways with major involvement of the alternative pathway.

Al(OH)3 is the most common adjuvant in human vaccines, but its mode of action remains poorly understood. Complement involvement in the adjuvant properties of Al(OH)3 has been suggested in several reports together with a depot effect. It is here confirmed that Al(OH)3 treatment of serum depletes complement components and activates the complement system. We show that complement activation by Al(OH)3 involves the three major pathways by monitoring complement components in Al(OH)3-treated serum and in Al(OH)3-containing precipitates. Al(OH)3 activation of complement results in deposition of C3 cleavage products and membrane attack complex (MAC) and in generation of the anaphylatoxins C3a and C5a. Complement activation was time dependent and inhibited by chelation with EDTA but not EGTA+Mg(2+). We thus confirm that Al(OH)3 activates the complement system and show that the alternative pathway is of major importance.

Complement factor B expression profile in a spontaneous uveitis model.

Equine recurrent uveitis serves as a spontaneous model for human autoimmune uveitis. Unpredictable relapses and ongoing inflammation in the eyes of diseased horses as well as in humans lead to destruction of the retina and finally result in blindness. However, the molecular mechanisms leading to inflammation and retinal degeneration are not well understood. An initial screening for differentially regulated proteins in sera of uveitic cases compared to healthy controls revealed an increase of the alternative pathway complement component factor B in ERU cases. To determine the activation status of the complement system, sera were subsequently examined for complement split products. We could demonstrate a significant higher concentration of the activation products B/Ba, B/Bb, Bb neoantigen, iC3b and C3d in uveitic condition compared to healthy controls, whereas for C5b-9 no differences were detected. Additionally, we investigated complement activation directly in the retina by immunohistochemistry, since it is the main target organ of this autoimmune disease. Interestingly, infiltrating cells co-expressed activated factor Bb neoantigen, complement split product C3d as well as CD68, a macrophage marker. In this study, we could demonstrate activation of the complement system both systemically as well as in the eye, the target organ of spontaneous recurrent uveitis. Based on these novel findings, we postulate a novel role for macrophages in connection with complement synthesis at the site of inflammation.

Induction of passive Heymann nephritis in complement component 6-deficient PVG rats.

Passive Heymann nephritis (PHN), a model of human membranous nephritis, is induced in susceptible rat strains by injection of heterologous antisera to rat renal tubular Ag extract. PHN is currently considered the archetypal complement-dependent form of nephritis, with the proteinuria resulting from sublytic glomerular epithelial cell injury induced by the complement membrane attack complex (MAC) of C5b-9. This study examined whether C6 and MAC are essential to the development of proteinuria in PHN by comparing the effect of injection of anti-Fx1A antisera into PVG rats deficient in C6 (PVG/C6(-)) and normal PVG rats (PVG/c). PVG/c and PVG/C6(-) rats developed similar levels of proteinuria at 3, 7, 14, and 28 days following injection of antisera. Isolated whole glomeruli showed similar deposition of rat Ig and C3 staining in PVG/c and PVG/C6(-) rats. C9 deposition was abundant in PVG/c but was not detected in PVG/C6(-) glomeruli, indicating C5b-9/MAC had not formed in PVG/C6(-) rats. There was also no difference in the glomerular cellular infiltrate of T cells and macrophages nor the size of glomerular basement membrane deposits measured on electron micrographs. To examine whether T cells effect injury, rats were depleted of CD8+ T cells which did not affect proteinuria in the early heterologous phase but prevented the increase in proteinuria associated with the later autologous phase. These studies showed proteinuria in PHN occurs without MAC and that other mechanisms, such as immune complex size, early complement components, CD4+ and CD8+ T cells, disrupt glomerular integrity and lead to proteinuria.

Dynamic expression of the membrane attack complex (MAC) of the complement system in failing human myocardium.

Inflammatory cytokine-mediated pathways are activated in heart failure and participate in the pathogenesis and progression of the disease. Another major response to inflammation is mediated through the complement system with the production of the membrane attack complex (MAC), a protein known to cause cell lysis and mediate apoptosis. It was postulated that the complement system is activated in patients with heart failure, and this study investigated whether hemodynamic conditions regulate this pathway. The expression of the MAC was assessed in myocardial biopsy samples of normal and failing hearts by immunohistochemistry and Western blot analysis. Myocardial samples from failing hearts were obtained before and after left ventricular assist device implantation. Immunohistochemical staining and Western blot analysis identified increased MAC expression in failing but not normal myocardium. After hemodynamic unloading with left ventricular assist device support, MAC expression returned to levels found in normal controls. In failing hearts, MAC expression did not differ between ischemic and nonischemic causes of heart failure. In conclusion, increased MAC expression in failing human hearts indicates that the complement system is activated in the heart failure milieu. Its removal after hemodynamic normalization is evidence of dynamic regulation, suggesting a pathogenic role for the MAC. These findings identify the complement system as part of a novel pathophysiologic path in heart failure that can potentially be targeted by future therapy.

Coagulation cascade activation causes CC chemokine receptor-2 gene expression and mononuclear cell activation in hemodialysis patients.

Priming of the coagulation cascade during hemodialysis (HD) leads to the release of activated factor X (FXa). The binding of FXa to its specific receptors, effector protease receptor-1 (EPR-1) and protease-activated receptor-2 (PAR-2), may induce the activation of peripheral blood mononuclear cells (PBMC) and promote a chronic inflammatory state that is responsible for several HD-related morbidities. In the attempt to elucidate the mechanisms underlying the coagulation-associated inflammation in HD, 10 HD patients were randomized to be treated subsequently with a cellulose acetate membrane (CA) and Ethylen-vinyl-alcohol (EVAL), a synthetic membrane that has been shown to reduce FXa generation. At the end of each experimental period, surface FXa and thrombin receptors (EPR-1 and PAR-1, -2, and -4) and CCR2 (monocyte chemoattractant protein-1 receptor) gene expression in isolated PBMC were examined. the ability of dialytic membranes to activate protein-tyrosine kinases and the stress-activated kinase JNK and to modulate the generation of terminal complement complex (TCC) was also investigated. EPR-1 and PAR-2 and -4 mRNA expression, barely detectable in normal PBMC, were significantly upregulated in HD patients, particularly in those who were treated with CA. A striking increase of tyrosine-phosphorylated proteins and JNK activation was observed at the end of HD only in CA-treated patients. Simultaneously, an increased gene expression for both splicing isoforms of CCR2, A and B, only in PBMC from CA-treated patients was demonstrated. The increased CCR-2 mRNA abundance was followed by a significant increase in its protein synthesis. The high expression of CCR2 was associated with an increased generation of plasma TCC and a significant drop in leukocyte and monocyte count. By contrast, EVAL treatment slightly lowered TCC generation and normalized leukocyte count. In vitro FXa induced CCR2 A and B expression and JNK activation in freshly isolated PBMC. FXa-induced CCR2 mRNA expression was completely abolished by JNK and tyrosine kinase inhibition. In conclusion, these data suggest that subclinical clotting activation may cause an increased CCR2 gene and protein expression on uremic PBMC, contributing to HD-related chronic microinflammation. The use of the less coagulation-activating membrane, EVAL, may reduce PBMC activation through the modulation of the stress-activated kinase JNK.

Neonatal porcine Sertoli cells inhibit human natural antibody-mediated lysis.

Sertoli cells protect cotransplanted cells from allogeneic and xenogeneic rejection. Additionally, neonatal porcine Sertoli cells (NPSCs) survive long-term as xenografts in nonimmunosuppressed rodents. This has led to the hypothesis that NPSCs could be used to prevent cellular rejection in clinical transplantation, thereby eliminating the need for chronic immunosuppression. Prior to transplantation of NPSCs in humans it is necessary to determine whether they are also protected from humoral-mediated xenograft rejection. The presence of Gal alpha(1,3)Gal beta(1,4)GlcNAc-R (alphaGal epitope) as well as binding of human immunoglobulin G (IgG) and IgM to NPSCs was examined by immunocytochemical and fluorescence-activated cell sorter analysis. alphaGal was detected on 88.5% +/- 3.0% of NPSCs. Consistent with this, 71.7% +/- 1.0% and 65.4% +/- 5.2% of NPSCs were bound by IgG and IgM, respectively. When cultured NPSCs underwent an in vitro cytotoxicity assay by incubation with human AB serum plus complement, no increase in cellular lysis was observed, while controls--porcine aorta endothelial cells--were shown to contain > 60% dead cells. Finally, activation of the complement cascade was examined by immunohistochemistry. C3 and C4 were deposited on the surface of the NPSC membrane, indicating activation of complement. Although the complement cascade was activated, the membrane attack complex (MAC) was not formed. These data demonstrate that despite expression of alphaGal, binding of xenoreactive antibodies, and the activation of complement, NPSCs survive human antibody and complement-mediated lysis by preventing MAC formation. This suggests that NPSCs may be able to survive humoral-mediated rejection in a clinical situation.

Soluble human complement receptor 1 limits ischemic damage in cardiac surgery patients at high risk requiring cardiopulmonary bypass.

BACKGROUND: This study was undertaken to determine whether soluble human complement receptor type 1 (TP10), a potent inhibitor of complement activation, would reduce morbidity and mortality in high-risk patients undergoing cardiac surgery on cardiopulmonary bypass (CPB). METHODS: This was a randomized multicenter, prospective, placebo-controlled, double-blind study in which 564 high-risk patients undergoing cardiac surgery on CPB received an intravenous bolus of TP10 (1, 3, 5, 10 mg/kg) or placebo immediately before CPB. The primary endpoint was the composite events of death, myocardial infarction (MI), prolonged (> or =24 hours) intra-aortic balloon pump support (IABP), and prolonged intubation. RESULTS: TP10 significantly inhibited complement activity after 10 to 15 minutes of CPB and this inhibition persisted for 3 days postoperatively. However, there was no difference in the primary endpoint between the 2 groups (33.7% placebo versus 31.4% TP10; P=0.31). The primary composite endpoint was, however, reduced in all male TP10 patients by 30% (P=0.025). TP10 reduced the incidence of death or MI in males by 36% (P=0.026), the incidence of death or MI in CABG males by 43% (P=0.043) and the need for prolonged IABP support in male CABG and valve patients by 100% (P=0.019). There was, however, no improvement seen in female TP10 patients. There were no significant differences in adverse events between the groups. CONCLUSIONS: TP10 effectively inhibits complement activation during CPB; however, this was not associated with an improvement in the primary endpoint of the study. Nevertheless, TP10 did significantly decrease the incidence of mortality and MI in male patients.

Complement activation in chromosome 13 dementias. Similarities with Alzheimer's disease.

Chromosome 13 dementias, familial British dementia (FBD) and familial Danish dementia (FDD), are associated with neurodegeneration and cerebrovascular amyloidosis, with striking neuropathological similarities to Alzheimer's disease (AD). Despite the structural differences among the amyloid subunits (ABri in FBD, ADan in FDD, and Abeta in AD), these disorders are all characterized by the presence of neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with markers of glial activation, suggestive of local inflammation. Proteins of the complement system and their pro-inflammatory activation products are among the inflammation markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain sections demonstrated the presence of complement activation components of the classical and alternative pathways as well as the neo-epitope of the membrane attack complex. Hemolytic experiments and enzyme-linked immunosorbent assays specific for the activation products iC3b, C4d, Bb, and C5b-9 indicated that ABri and ADan are able to fully activate the complement cascade at levels comparable to those generated by Abeta1-42. ABri and ADan specifically bound C1q with high affinity and formed stable complexes in physiological conditions. Activation proceeds approximately 70-75% through the classical pathway while only approximately 25-30% seems to occur through the alternative pathway. The data suggest that the chronic inflammatory response generated by the amyloid peptides in vivo might be a contributing factor for the pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative conditions.

The inhibitory effect of CD46, CD55, and CD59 on complement activation after immunotherapeutic treatment of cervical carcinoma cells with monoclonal antibodies or bispecific monoclonal antibodies.

The role of membrane-bound complement regulatory proteins (mCRP) in the protection of tumor cells in vivo against elimination by the immune system is still unknown. In this study the effect of expression of these mCRP by cervical cancer cells was investigated. In situ expression of mCRP was observed on cervical carcinomas, normal cervical epithelial cells, and the surrounding stroma. Deposition of C3 and C5b-9 was sporadically found on the tumor cells and the surrounding stroma. A low expression of CD46 was statistically significantly associated with deposition of C3. Comparable expression patterns were shown on primary cervical tumor cell suspensions. A relatively high deposition of C4c was found on these tumor cells, indicating classical pathway activation. Furthermore, it was demonstrated that CD55 and CD59 were the most potent inhibitors of C3 deposition and classical pathway-mediated lysis, respectively, on cervical cancer cell lines. The feasibility of increasing complement activation at the tumor cell membrane surface was demonstrated with an anti-HLA Class I*anti-CD55 bispecific mAb. The potential immunotherapeutic applicability was investigated with both anti-G250*anti-CD55 and anti-Ep-CAM*anti-CD55 bispecific mAbs. An approximate 2-fold increase in C3 deposition, compared with the parental anti-Ep-CAM mAb, was attained with an anti-Ep-CAM*anti-CD55 bispecific mAb when the tumor-associated antigen was expressed in sufficient amounts. These results demonstrate that when tumor-associated antigens are expressed in adequate amounts, bispecific mAbs in vivo may be potent immunotherapeutic agents to enhance an inflammatory reaction at the tumor site.

Intraglomerular synthesis of complement C3 and its activation products in IgA nephropathy.

BACKGROUND: Complement activation is thought to be pathologically important in IgA nephropathy (IgAN). Although C3 deposition in the mesangium is found in IgAN, the origin of C3 is not clear. We recently demonstrated intraglomerular C3 synthesis in the human kidney; however, the activation and pathological role of locally synthesized C3 remains unclear. Here we performed nonradioactive in situ hybridization for C3 mRNA and immunohistochemistry for C3 and its activation products, such as C3d and membrane attack complex (MAC), to determine whether locally produced C3 in glomeruli was activated in IgA nephropathy. METHODS: Renal samples from 14 patients with IgAN and 5 with minimal change nephrotic syndrome (MCNS) were examined. Uninvolved portions of surgically removed kidneys with tumors served as normal controls. RESULTS: C3 mRNA was not detected in glomeruli in control tissue and MCNS, but was strongly expressed in resident glomerular cells of IgAN, including mesangial cells, glomerular epithelial cells and the cells of Bowman's capsule. Examination of serial sections disclosed that more than 70% of cells positive for C3 mRNA were also stained for C3 protein, C3d, and MAC. Double staining for in situ hybridization and immunohistochemistry also revealed that those C3 mRNA signals were present in intraglomerular cells positive for C3. The expression of C3 mRNA and MAC in glomeruli correlated significantly with the degree of mesangial matrix expansion. CONCLUSIONS: Our results demonstrated that locally synthesized C3 is activated in the glomeruli of IgAN and that its expression correlated with the severity of mesangial matrix expansion. These findings suggest that activation of C3 may be involved in tissue injury in IgAN through the formation of membrane attack complex.

Biochemical background of paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by paroxysms of intravascular hemolysis. A considerable part of erythrocytes in patient blood is susceptible to autologous complement activation because of the deficiency of CD59, which is a glycosylphosphatidylinositol (GPI)-anchored protein and inhibits the formation of the membrane attack complex (MAC) of complement. The deficiency of CD59 is derived from the inability of GPI-anchor synthesis. Although more than 10 proteins are involved in the GPI-anchor synthesis, the mutation of only one protein, PIG-A, causes the defect in about 200 patients with PNH who have been analyzed. The reason why only PIG-A causes the deficiency of GPI anchor is due to the location of its gene on X chromosome. The clonal stem cell mutated with PIG-A gene in the bone marrow loses the capability of the synthesis of GPI-anchor. The mutation of PIG-A gene alone, however, seems to be insufficient to account for the survival of the PIG-A-deficient cells in the bone marrow. Thus, a fraction of the mutant stem cells probably gain a survival advantage by some additional changes, either additional mutations or changes in immunological circumstances. The release of the surviving cells into blood stream results in a clinical syndrome with PNH.

A new in vitro model to study interaction between whole blood and biomaterials. Studies of platelet and coagulation activation and the effect of aspirin.

We have developed a versatile in vitro chamber model with a double purpose: first, to be able to study mechanisms of bio-incompatibility, and, second, to test biomaterials at all levels of interactions, in whole blood. The use of biomaterials in the form of microscope slides as walls in the chamber makes it possible to analyse both the biomaterial surface with regard to protein and cell binding, as well as the molecular events taking place in the fluid. Incubation of blood in the chamber, for 60 min at 37 degrees C resulted in the rapid binding of complement and coagulation proteins and of leukocytes and platelets to polyvinylchloride (PVC) slides. The cells formed a layer which more or less covered the underlying surface. Unlike complement activation, as reflected by soluble C3a and C5b-9, the thrombin-antithrombin formation was completely nullified in cell-depleted plasma. Despite the fact that thrombin-antithrombin generation was also negligible in platelet-rich plasma, inhibition of platelet aggregation on the material surface with aspirin resulted in suppressed generation of thrombin antithrombin complexes. Taken together, the coagulation activation in the chamber was dependent on the presence of blood cells which suggests that bound/aggregated platelets initiate a sequence of events involving leukocytes that results in coagulation activation.

In vivo generation of C4d, Bb, iC3b, and SC5b-9 after OKT3 administration in kidney and lung transplant recipients.

BACKGROUND: OKT3 monoclonal antibody therapy results in an acute clinical syndrome (ACS) associated with the release of tumor necrosis factor and sequestration of neutrophils in the lungs. We have previously shown that inhibition of tumor necrosis factor does not completely eliminate OKT3-ACS, suggesting that other factors also contribute to the ACS. The current studies analyzed complement activation in vivo during the first hour after OKT3 administration. METHODS: Renal (n=4) and lung (n=4) transplant recipients received OKT3 as treatment for rejection and induction therapy, respectively. Complement activation products C4d, Bb, iC3b, and SC5b-9 were measured by ELISA. Hemodynamic parameters were also monitored in the lung transplant recipients. Neutrophil expression of CD11a, CD11b, and CD18 was monitored by flow cytometry. Controls included patients receiving methylprednisolone for rejection (n=4), two adults with adult respiratory distress syndrome who received extracorporeal membrane oxygenation, and normal volunteers (n=5). P values less than 0.05 (*) were considered significant. RESULTS: Increases in the plasma levels of C4d, Bb, iC3b, and SC5b-9 were observed in seven of eight patients after OKT3 administration. Mean values (n=8) at 0, 15, and 60 min (in microg/ml) were as follows-C4d: 1.865, 2.644*, and 2.607*; Bb: 0.245, 0.411, and 0.385; iC3b: 10.881, 17.242*, and 15.145*; and SC5b-9: 0.232, 0.269, and 0.302*. An increase in CD11b and CD18 and a decrease of CD11a on neutrophils in parallel with complement activation was observed. In lung transplant recipients, C3 activation correlated with increases in mean pulmonary and central venous pressures (P<0.05). As compared with extracorporeal membrane oxygenation, which activated classical and alternative pathways, OKT3 predominantly activated complement by the classical pathway. Methylprednisolone pulses did not activate complement. CONCLUSIONS: Complement activation is an early event after OKT3 administration and is associated with the increased expression of adhesion molecules on neutrophils and with pulmonary hemodynamic changes. Effective therapeutic approaches to the control of early monoclonal antibody side effects may require measures that limit complement activation in addition to reducing cytokine activity.

Interleukin-1 alpha, interleukin 6 and tumor necrosis factor alpha increase the synthesis and expression of the functional alternative and terminal complement pathways by human umbilical vein endothelial cells in vitro.

The proinflammatory cytokines interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF alpha) and interleukin 6 (IL-6) modulate the synthesis of complement factors B and C3 by endothelial cells (EC), and are considered to play an important role in the development of sepsis. By using agarose beads activating the alternative pathway of complement, we wanted to study the net effect of these cytokines on EC synthesis of the alternative and terminal pathways, measured by binding of anti-C3c and anti-TCC (terminal complement complex) antibodies to beads kept with the EC. Addition of IL-1 alpha and TNF alpha at concentrations of 50 and 100 U/ml resulted in a significant increase in binding of these antibodies to co-incubated beads, most pronounced for anti-C3c. IL-6 from 50-200 U/ml resulted in a stronger (two to fourfold) binding for both antibodies compared to experiments with IL-1 alpha and TNF. However, increased concentrations of IL-1 alpha (200 U/ml) and IL-6 (400 U/ml) resulted in a strong reduction in binding of anti-C3c and anti-TCC antibodies to the co-cultured beads. This study indicates that proinflammatory cytokines upregulate the synthesis by EC of the functional alternative and terminal pathways of complement.

Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines.

C biosynthesis at extrahepatic sites remote from plasma C may be important in the protection of tissues against inflammation and infection but may also contribute to tissue injury. This latter possibility is particularly relevant in the central nervous system (CNS), where several cell types are susceptible to damage by C. We have previously shown that human astrocyte-derived tumor cell lines synthesize and secrete all of the components of the activation pathways of C. In this study, we demonstrate that these cells also produce the components (C6, C7, C8, and C9) and regulators (S-protein and clusterin) of the lytic terminal C pathway. The terminal components produced are hemolytically active, and secretion is markedly up-regulated by the inflammatory cytokine IFN-gamma. Primary human fetal astrocytes also expressed C6, C7, S-protein, and clusterin. The human monocyte/macrophage cell line, used here as a model for microglia, also produced all terminal components and regulators when appropriately stimulated. These studies raise the prospect of the intrathecal synthesis of a complete, functional C system and its regulators in the inflamed CNS. Intrathecal C synthesis may be important in the resolution of infection and inflammation but, given the C susceptibility of some CNS cell types, may also exacerbate damage in demyelination and neurodegeneration.

Complement activation during cardiopulmonary bypass: effects of immobilized heparin.

The role of complement in biocompatibility reactions and the correlation between complement activation during cardiopulmonary bypass (CPB) and postperfusion syndrome have inspired attempts to improve the biocompatibility of extracorporeal blood oxygenation devices. Here we assessed the effect of immobilized heparin on the generation of C3a and terminal complement complexes during CPB. Thirty patients undergoing aortocoronary bypass were randomized to CPB with either heparin-coated (Duraflo II; Bentley, Irvine, CA) or noncoated control membrane oxygenators (Univox; Bentley). A standard dose of heparin (300 IU/kg) was given to the control group while the heparin dose was reduced to 30% (100 IU/kg) in the heparin-coated group. Significantly lower levels of terminal complement complexes were detected in the heparin-coated group by the end of CPB. From 28 +/- 5 AU/mL (heparin-coated group) and 26 +/- 3 AU/mL (control group, mean +/- standard error of the mean) the terminal complement complex levels increased to 391 +/- 35 AU/mL and 602 +/- 47 AU/mL, respectively (p < 0.002). This difference was still apparent 180 minutes after CPB. Although there was no difference in C3a levels between the two groups at the end of CPB, C3a levels were significantly lower in the heparin-coated group 30 minutes after CPB (194 +/- 18 ng/mL and 307 +/- 18 ng/mL in heparin-coated and control groups, respectively; p < 0.001). We conclude that the heparin-coated surface is more biocompatible with regard to complement activation than is the ordinary unmodified surface in extracorporeal circuits.

Cell-surface bound complement regulatory activity is necessary for the in vivo survival of KDH-8 rat hepatoma.

The monoclonal antibody 5I2 recognizes and functionally inhibits a Crry-like complement regulator molecule on rat cells (corresponding to human decay accelerating factor and membrane cofactor protein activity). The inhibition of complement regulatory activity by 5I2 antibody results in complement deposition on rat cell membranes exposed to homologous complement. Two subclones of KDH-8 rat hepatoma were selected for the experiments; one expressing high (CrryH) and the second low (CrryL) amounts of Crry-like antigen. Both sublines grow in vivo in syngeneic rats, but at lower cell doses (< 10(5) cells/rat) the survival time of rats injected with CrryL cells is prolonged. Injection of tumour-bearing rats with 5I2 monoclonal antibody intraperitoneally did not influence the tumour growth. However, it resulted in 50% mortality within a few hours, accompanied by severe haemorrhage in the peritoneal cavity. Pretreatment of the tumour cells with 5I2 monoclonal antibody or its F(ab')2 fragment substantially increased the survival time of recipient rats. Even permanent survivors were found, indicating that cell membrane-associated complement regulatory activity, which provides protection against attack of homologous complement, is necessary for in vivo tumour growth.