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Background: Burns induce a boost in local and systemic complement levels as well as immune cell infiltration in the burn wound, that may negatively affect wound healing. Objective: In this study, the effects of long-term treatment with complement inhibitor C1 esterase inhibitor (C1inh) on post-burn inflammation and wound healing parameters were analyzed in time up to 60 days post-burn. Method: Burned pigs were treated either with or without C1inh up to 15 days post-burn. Burn wound biopsies and blood were collected at different time points up to 60 days post-burn. Thereafter, complement in blood as well as complement and immune cells in the wound, capillary leakage, necrosis, reepithelialization and wound contraction were quantified. Results: No significant differences in complement C3 blood levels were observed at any time point between C1inh-treated and control pigs. In the wound, complement C4 levels were significantly lower in the C1inh group than in controls at day 3-6 and 21-30 post-burn. Similarly, C3 levels, neutrophil and macrophage infiltration in the wound were, although not statistically significant, reduced in C1inh-treated pigs at day 9-14 post-burn. No differences in lymphocyte infiltration in the wound were found between C1inh and control pigs. C1inh-treated pigs also showed reduced capillary leakage. Despite these effects, no significant differences in the long-term wound healing parameters necrosis, reepithelialization and wound contraction were observed between C1inh and control pigs. Conclusion:: In pigs 15 days of C1inh treatment after burn, leads to a reduction in local inflammation and capillary leakage in the burn wound without affecting long-term wound healing parameters.

Background C1-inhibitor (C1-inh) therapeutics can reduce neutrophil activity in various inflammatory conditions. This ‘novel’ anti-inflammatory effect of C1-inh is attributed to the tetrasaccharide sialyl LewisX (SLeX) present on its N-glycans. Via SLeX, C1-inh is suggested to interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain plasma glycoprotein α1-antichymotrypsin (ACT) as a co-purified protein impurity. Objective This article investigates the contribution of ACT to the effects observed with C1-inh. Materials and Methods We have separated C1-inh and ACT from a therapeutic C1-inh preparation and investigated the influence of these proteins on SLeX–selectin interactions in a specific in vitro model, which makes use of rolling of SLeX-coated beads on immobilized E-selectin. Results We find that ACT and not C1-inh, shows a clear sialic acid-dependent interference in SLeX–selectin interactions, at concentrations present in C1-inh therapeutics. Furthermore, we do not find any evidence of SLeX on C1-inh using either Western blotting with anti-SLeX antibodies (CSLEX1 and KM93) or by mass spectrometric analysis of N-glycans. C1-inh reacts weakly to antibody HECA-452, which detects a broad range of selectin ligands, but ACT gives a much stronger signal, suggesting the presence of a selectin ligand on ACT. Conclusion The ‘novel’ anti-inflammatory effects of C1-inh are unlikely due to SLeX on C1-inh and can in fact be due to SLeX-like glycans on ACT, present in C1-inh products. In view of our results, it is important to assess the role of ACT in vivo and revisit past studies performed with commercial C1-inh.

The complement system plays an important role in the inflammatory response subsequent to acute myocardial infarction (AMI). The aim of this study is to create a systematic overview of studies that have investigated therapeutic administration of complement inhibitors in both AMI animal models and human clinical trials. To enable extrapolation of observations from included animal studies toward post-AMI clinical trials, ex vivo studies on isolated hearts and proof-of-principle studies on inhibitor administration before experimental AMI induction were excluded. Positive therapeutic effects in AMI animal models have been described for cobra venom factor, soluble complement receptor 1, C1-esterase inhibitor (C1-inh), FUT-175, C1s-inhibitor, anti-C5, ADC-1004, clusterin, and glycosaminoglycans. Two types of complement inhibitors have been tested in clinical trials, being C1-inh and anti-C5. Pexelizumab (anti-C5) did not result in reproducible beneficial effects for AMI patients. Beneficial effects were reported in AMI patients for C1-inhibitor, albeit in small patient groups. In general, despite the absence of consistent positive effects in clinical trials thus far, the complement system remains a potentially interesting target for therapy in AMI patients. Based on the study designs of previous animal studies and clinical trials, we discuss several issues which require attention in the design of future studies: adjustment of clinical trial design to precise mechanism of action of administered inhibitor, optimizing the duration of therapy, and optimization of time point(s) on which therapeutic effects will be evaluated.

Mutations in the gene encoding for complement regulator factor H (FH) severely disrupt its normal function to protect human cells from unwanted complement activation, resulting in diseases such as atypical hemolytic uremic syndrome (aHUS). aHUS presents with severe hemolytic anemia, thrombocytopenia, and renal disease, leading to end-stage renal failure. Treatment of severe complement-mediated disease, such as aHUS, by inhibiting the terminal complement pathway, has proven to be successful but at the same time fails to preserve the protective role of complement against pathogens. To improve complement regulation on human cells without interfering with antimicrobial activity, we identified an anti-FH monoclonal antibody (mAb) that induced increased FH-mediated protection of primary human endothelial cells from complement, while preserving the complement-mediated killing of bacteria. Moreover, this FH-activating mAb restored complement regulation in sera from aHUS patients carrying various heterozygous mutations in FH known to impair FH function and dysregulate complement activation. Our data suggest that FH normally circulates in a less active conformation and can become more active, allowing enhanced complement regulation on human cells. Antibody-mediated potentiation of FH may serve as a highly effective approach to inhibit unwanted complement activation on human cells in a wide range of hematological diseases while preserving the protective role of complement against pathogens. ispartof: BLOOD ADVANCES vol:3 issue:4 pages:621-632 ispartof: location:United States status: published

The lectin pathway (LP) of complement activation depends on the activation of the MBL-associated serine proteases (MASPs) circulating in complex with mannan-binding lectin (MBL). MBL deficiency is the most common complement deficiency and has been associated with several pathological conditions. As we had previously shown, plasma-derived MBL (pdMBL) contains pre-activated MASPs that upon in vivo pdMBL substitution results in restoration of MBL concentrations but no LP functionality due to immediate inactivation of pdMBL-MASP complexes upon infusion. In this study, we analyzed MBL-sufficient and -deficient serum by size-exclusion chromatography for complexes of LP activation. In both sera, we identified non-bound free forms of MASP-2 and to lesser extent MASP-1/3. After addition of recombinant MBL (rMBL) to MBL-deficient serum, these free MASPs were much less abundantly present, which is highly suggestive for the formation of high-molecular complexes that could still become activated upon subsequent ligand binding as shown by a restoration of C4-deposition of MBL-deficient serum. Ficolin (FCN)-associated MASPs have been described to redistribute to ligand-bound MBL, hereby forming new MBL/MASP complexes. However, reconstitution of MBL-deficient serum with rMBL did not change the relative size of the FCN molecules suggestive for a limited redistribution in fluid phase of already formed complexes. Our findings demonstrate that rMBL can associate with free non-bound MASPs in fluid phase while preserving full restoration of LP functionality. In contrast to pdMBL products containing pre-activated MASPs which become inactivated almost immediately, these current data provide a rationale for substitution studies using rMBL instead.

Background Complement activation contributes significantly to inflammation-related damage in the heart after acute myocardial infarction. Knowledge on factors that regulate postinfraction complement activation is incomplete however. In this study, we investigated whether endogenous C1-inhibitor, a well-known inhibitor of complement activation, is expressed in the heart after acute myocardial infarction. Materials and methods C1-inhibitor and complement activation products C3d and C4d were analyzed immunohistochemically in the hearts of patients who died at different time intervals after acute myocardial infarction ( n = 28) and of control patients ( n = 8). To determine putative local C1-inhibitor production, cardiac transcript levels of the C1-inhibitor-encoding gene serping1 were determined in rats after induction of acute myocardial infarction (microarray). Additionally, C1-inhibitor expression was analyzed (fluorescence microscopy) in human endothelial cells and rat cardiomyoblasts in vitro. Results C1-inhibitor was found predominantly in and on jeopardized cardiomyocytes in necrotic infarct cores between 12 h and 5 days old. C1-inhibitor protein expression coincided in time and colocalized with C3d and C4d. In the rat heart, serping1 transcript levels were increased from 2 h up until 7 days after acute myocardial infarction. Both endothelial cells and cardiomyoblasts showed increased intracellular expression of C1-inhibitor in response to ischemia in vitro ( n = 4). Conclusions These observations suggest that endogenous C1-inhibitor is likely involved in the regulation of complement activity in the myocardium following acute myocardial infarction. Observations in rat and in vitro suggest that C1-inhibitor is produced locally in the heart after acute myocardial infarction.

Human C1-inhibitor (C1-Inh) is a serine protease inhibitor and the major regulator of the contact activation pathway as well as the classical and lectin complement pathways. It is known to be a highly glycosylated plasma glycoprotein. However, both the structural features and biological role of C1-Inh glycosylation are largely unknown. Here, we performed for the first time an in-depth site-specific N- and O-glycosylation analysis of C1-Inh combining various mass spectrometric approaches, including C18-porous graphitized carbon (PGC)-LC-ESI-QTOF-MS/MS applying stepping-energy collision-induced dissociation (CID) and electron-transfer dissociation (ETD). Various proteases were applied, partly in combination with PNGase F and exoglycosidase treatment, in order to analyze the (glyco)peptides. The analysis revealed an extensively O-glycosylated N-terminal region. Five novel and five known O-glycosylation sites were identified, carrying mainly core1-type O-glycans. In addition, we detected a heavily O-glycosylated portion spanning from Thr82-Ser121 with up to 16 O-glycans attached. Likewise, all known six N-glycosylation sites were covered and confirmed by this site-specific glycosylation analysis. The glycoforms were in accordance with results on released N-glycans by MALDI-TOF/TOF-MS/MS. The comprehensive characterization of C1-Inh glycosylation described in this study will form the basis for further functional studies on the role of these glycan modifications.

In autoimmune hemolytic anemia autoantibodies against erythrocytes lead to increased clearance of the erythrocytes, which in turn results in a potentially fatal hemolytic anemia. Depending on whether IgG or IgM antibodies are involved, response to therapy is different. Proper identification of the isotype of the anti-erythrocyte autoantibodies is, therefore, crucial. However, detection of IgM autoantibodies can be challenging. We, therefore, set out to improve the detection of anti-erythrocyte IgM. Direct detection using a flow cytometry-based approach did not yield satisfactory improvements. Next, we analyzed whether the presence of complement C3 on a patient’s erythrocytes could be used for indirect detection of anti-erythrocyte IgM. To this end, we fractionated patients’ sera by size exclusion chromatography and tested which fractions yielded complement deposition on erythrocytes. Strikingly, we found that all patients with C3 on their erythrocytes according to standard diagnostic tests had an IgM anti-erythrocyte component that could activate complement, even if no such autoantibody had been detected with any other test. This also included all tested patients with only IgG and C3 on their erythrocytes, who would previously have been classified as having an IgG-only mediated autoimmune hemolytic anemia. Depleting patients’ sera of either IgG or IgM and testing the remaining complement activation confirmed this result. In conclusion, complement activation in autoimmune hemolytic anemia is mostly IgM-mediated and the presence of covalent C3 on patients’ erythrocytes can be taken as a footprint of the presence of anti-erythrocyte IgM. Based on this finding, we propose a diagnostic workflow that will aid in choosing the optimal treatment strategy.

Autoimmune hemolytic anemia (AIHA) is a rare autoimmune disease characterized by a hemolytic anemia caused by autoantibodies against red blood cells (RBCs). These autoantibodies are routinely detected via the direct antiglobulin test (DAT). As expected, the DAT score correlates with the presence of clinical symptoms, but this correlation is far from perfect. Regularly, strongly positive DAT scores are encountered with no sign of hemolysis, while severe hemolysis can be seen even in patients with a negative DAT score. Apparently, the mere amount of antibody is not the sole predictor of disease. In this paper, we review the current literature on aspects of both the autoantibodies and the patients' clearance system that together determine the clinical significance of an anti-RBC autoantibody, ranging from antibody isotype to antibody Fc-glycosylation to alternative clearance mechanisms. From this, the ensemble of tests is inferred that in our view best assesses the main determinants for pathogenicity of autoantibodies.

Background Neutralizing autoantibodies (NAbs) against plasma serpin C1-inhibitor (C1-inh) are implicated in the rare disorder, acquired angioedema (AAE). There is insufficient understanding of the process of antibody formation and its correlation with disease progression and severity. We have developed an ELISA for detecting neutralizing capacity of anti-C1-inh positive plasma samples that can be used to study changes in NAb repertoire in patient plasma over the course of disease. Methods The ELISA is based on the specific interaction of active C1-inh with its target protease C1s. Decrease in the amount of C1s bound to immobilized C1-inh in the presence of test samples is proportional to the neutralizing capacity of the sample. Assay specificity, intra- and inter-assay variation and assay cut-off are determined using anti-C1-inh antibodies. Assay capability is demonstrated using plasma samples from AAE patients. Results The assay is specific to a neutralizing anti-C1-inh antibody and shows no interference by a non-neutralizing anti-C1-inh antibody or by the plasma matrix. Intra-assay and inter-assay variations are determined as 17 and 18% respectively. Neutralizing capacity of antibody positive AAE patient plasma samples (n = 16) with IgG or IgM type antibodies is readily determined. All samples show positive neutralizing capacity. Conclusion We have developed a robust, specific and semi-quantitative assay to detect the neutralizing capacity of plasma samples containing anti-C1-inh antibodies. This assay can be an important tool for the study of clinical implications of anti-C1-inh NAbs.

After mucosal damage or gingival inflammation, complement proteins leak into the oral cavity and mix with salivary proteins such as salivary agglutinin (SAG/gp-340/DMBT1). This protein is encoded by the gene Deleted in Malignant Brain Tumors 1 (DMBT1), and it aggregates bacteria, viruses and fungi, and activates the lectin pathway of the complement system. In the lectin pathway, carbohydrate structures on pathogens or altered self cells are recognized. SAG is highly glycosylated, partly on the basis of the donor’s blood group status. Whereas secretors express Lewis b, Lewis y, and antigens from the ABO-blood group system on SAG, non-secretors do not. Through mannose-binding lectin (MBL) binding and C4 deposition assays, we aimed to identify the chemical structures on SAG that are responsible for complement activation. The complement-activating properties of SAG were completely abolished by oxidation of its carbohydrate moiety. SAG-mediated activation of complement was also inhibited in the presence of saccharides such as fucose and Lewis b carbohydrates, and also after pretreatment with the fucose-binding lectin, Anguilla anguilla agglutinin. Complement activation was significantly (p

Complement activation in autoimmune hemolytic anemia may exacerbate extravascular hemolysis and may occasionally result in intravascular hemolysis. IgM autoantibodies as characteristically found in cold autoantibody autoimmune hemolytic anemia, in cold agglutinin disease but also in a considerable percentage of patients with warm autoantibodies are very likely to activate complement in vivo. Therapy of IgM-mediated autoimmune hemolytic anemia mainly aims to decrease autoantibody production. However, most of these treatments require time to become effective and will not stop immediate ongoing complement-mediated hemolysis nor prevent hemolysis of transfused red blood cells. Therefore pharmacological inhibition of the complement system might be a suitable approach to halt or at least attenuate ongoing hemolysis and improve the recovery of red blood cell transfusion in autoimmune hemolytic anemia. In recent years, several complement inhibitors have become available in the clinic, some of them with proven efficacy in autoimmune hemolytic anemia. In the present review, we give a short introduction on the pathogenesis of autoimmune hemolytic anemia, followed by an overview on the complement system with a special focus on its regulation. Finally, we will discuss complement inhibitors with regard to their potential efficacy to halt or attenuate hemolysis in complement-mediated autoimmune hemolytic anemia.

The complement factor H-related (FHR) proteins are hypothesized to fine-tune the regulatory role of complement factor H (FH) in the alternative pathway of the complement system. Moreover, FHR-1, FHR-2, and FHR-5 have been proposed to be dimers, which further complicates accurate analysis. As FHRs are highly similar among themselves and toward FH, obtaining specific reagents for quantification of serum levels and functional analysis is challenging. In this study, we generated antibodies and developed ELISAs to measure FHR-1, FHR-2, and FHR-5 in serum. We used both recombinant and serum-derived proteins to show that four dimers occur in human circulation: homodimers of FHR-1, FHR-2, and FHR-5, as well as FHR-1/FHR-2 heterodimers. Heterodimers containing FHR-5 were not found. In individuals with homozygous; CFHR1; deletions or compound heterozygous; CFHR2; missense/nonsense mutations identified in this study, the respective FHR-1 and FHR-2 homo- and heterodimers were absent. Using FRET, we found that recombinant FHR dimers exchange monomers rapidly. This was confirmed; ex vivo; , using FHR-1- and FHR-2-deficient sera. Of all FHR dimers, FHR-5/5 homodimers demonstrated strong binding affinity toward heparin. Specific ELISAs demonstrated that serum levels of FHR-1/1, FHR-1/2, FHR-2/2, and FHR-5/5 dimers were low compared to FH, which circulates at a 10- to 200-fold molar excess. In summary, FHR-1, FHR-2, and FHR-5 homodimerize, with FHR-1 and FHR-2 forming heterodimers as well, and equilibrate quickly in plasma.

Objective. The aim was to investigate the association between autoantibodies (autoAbs) and neuropsychiatric (NP) involvement in patients with SLE and to evaluate whether any autoAb or a combination of these autoAbs could indicate the underlying pathogenic process. Methods. Using a multiplexed protein array for 94 antigens, we compared the serum autoAb profiles of 69 NPSLE patients, 203 SLE patients without NP involvement (non-NPSLE) and 51 healthy controls. Furthermore, we compared the profiles of NPSLE patients with clinical inflammatory (n = 38) and ischaemic (n = 31) NP involvement. Results. In total, 75 IgG and 47 IgM autoAbs were associated with SLE patients in comparison with healthy controls. Comparing NPSLE with non-NPSLE and healthy control sera, 9 IgG (amyloid, cardiolipin, glycoprotein 2, glycoprotein 210, heparin, heparan sulphate, histone H2A, prothrombin protein and vimentin) and 12 IgM (amyloid, cardiolipin, centromere protein A, collagen II, histones H2A and H2B, heparan sulphate, heparin, mitochondrial 2, nuclear Mi-2, nucleoporin 62 and vimentin) autoAbs were present at significantly different levels in NPSLE. The combination of IgG autoAbs against heparan sulphate, histone H2B and vimentin could differentiate NPSLE from non-NPSLE (area under the curve 0.845, 99.97% CI: 0.756, 0.933; P < 0.0001). Compared with non-NPSLE, four IgG and seven IgM autoAbs were significantly associated with inflammatory NPSLE. In ischaemic NPSLE, three IgG and three IgM autoAbs were significantly different from non-NPSLE patients. Conclusion. In our cohort, the presence of high levels of anti-heparan sulphate and anti-histone H2B combined with low levels of anti-vimentin IgG autoAbs is highly suggestive of NPSLE. These results need to be validated in external cohorts.

Glycosylation of the immunoglobulin G (IgG)-Fc tail is required for binding to Fc-gamma receptors (FcγRs) and complement-component C1q. A variety of IgG1-glycoforms is detected in human sera. Several groups have found global or antigen-specific skewing of IgG glycosylation, for example in autoimmune diseases, viral infections, and alloimmune reactions. The IgG glycoprofiles seem to correlate with disease outcome. Additionally, IgG-glycan composition contributes significantly to Ig-based therapies, as for example IVIg in autoimmune diseases and therapeutic antibodies for cancer treatment. The effect of the different glycan modifications, especially of fucosylation, has been studied before. However, the contribution of the 20 individual IgG glycoforms, in which the combined effect of all 4 modifications, to the IgG function has never been investigated. Here, we combined six glyco-engineering methods to generate all 20 major human IgG1-glycoforms and screened their functional capacity for FcγR and complement activity. Bisection had no effect on FcγR or C1q-binding, and sialylation had no- or little effect on FcγR binding. We confirmed that hypo-fucosylation of IgG1 increased binding to FcγRIIIa and FcγRIIIb by ~17-fold, but in addition we showed that this effect could be further increased to ~40-fold for FcγRIIIa upon simultaneous hypo-fucosylation and hyper-galactosylation, resulting in enhanced NK cell-mediated antibody-dependent cellular cytotoxicity. Moreover, elevated galactosylation and sialylation significantly increased (independent of fucosylation) C1q-binding, downstream complement deposition, and cytotoxicity. In conclusion, fucosylation and galactosylation are primary mediators of functional changes in IgG for FcγR- and complement-mediated effector functions, respectively, with galactose having an auxiliary role for FcγRIII-mediated functions. This knowledge could be used not only for glycan profiling of clinically important (antigen-specific) IgG but also to optimize therapeutic antibody applications.

BACKGROUND: Diagnosing lymphocytic myocarditis (LM) is challenging because of the large variation in clinical presentation and the limitations inherent in current diagnostic tools. The objective of this study was to analyze infiltration of inflammatory cells in quadriceps skeletal muscle of LM patients and investigate the potential diagnostic value of assaying infiltrating inflammatory cells.METHODS: Quadriceps muscle tissue, obtained at autopsy from control patients (n = 9) and LM patients (n = 21), was analyzed using immunohistochemistry for infiltration of lymphocytes (CD45), macrophages (CD68), neutrophilic granulocytes (myeloperoxidase), and several lymphocyte subtypes (CD3, CD4, CD8, CD20) and using polymerase chain reaction for a panel of myocarditis-associated viruses. Additionally, quadriceps muscle from mice with acute coxsackievirus B3-induced myocarditis and control mice was analyzed for presence of lymphocytes and virus.RESULTS: In quadriceps muscle of LM patients the number of infiltrating lymphocytes were significantly increased and LM was diagnosed with specificity of 100% and sensitivity of 71%. Parvovirus B19 was the primary virus found in our patient groups, found in quadriceps tissue of 3 LM patients (although it was also found in 1 control patient). In the mice, enteroviral RNA was present in the quadriceps muscle, although enteroviral capsid proteins and lymphocyte infiltration were found primarily in the adipose tissue within and directly adjacent to the myocyte tissue, rather than in the myocyte tissue itself.CONCLUSIONS: LM is associated with lymphocyte infiltration and viral presence in quadriceps muscle. This indicates that skeletal muscle biopsy/lymphocyte quantification might be a potential diagnostic tool for LM patients.

Objective In rheumatoid arthritis (RA), autoantibodies such as anti–citrullinated protein antibodies (ACPAs) develop in response to neoepitopes that are formed under conditions of chronic inflammation. These autoantibodies may subsequently be fragmented by inflammation-associated proteases, leading to the formation of F(ab′)2 fragments. The hinge of F(ab′)2 fragments can serve as a neoepitope, and so-called antihinge antibodies (AHAs) can be found in RA patients, which might modulate the function of (fragmented) autoantibodies. We undertook this study to investigate the presence and specificities of AHAs in different stages of RA and to study their function. Methods The presence of AHAs was assessed by radioimmunoassay in healthy controls, blood donors who later developed RA, patients with arthralgia, patients with early RA, and patients with established RA. Specificity of the AHAs was analyzed with inhibition assays, and complement-activating ability was studied with a C4b deposition assay. Results Antibodies to IgG1 hinge, IgG2 hinge, and IgG4 hinge were detected in patients with established RA, with anti–IgG4 hinge antibodies being most specific (appearing in 1% of healthy controls, 3.8% of blood donors who later developed RA, 13% of arthralgia patients, 19% of early RA patients, and 16% of established RA patients). Anti–IgG4 hinge antibodies were subclass specific and were able to restore C4b deposition by IgG4 F(ab′)2 fragments. In patients with arthralgia and patients with early RA, anti–IgG4 hinge antibodies were associated with rheumatoid factors and ACPAs. Conclusion Anti–IgG4 hinge antibodies are present in RA patients and have low sensitivity but high specificity for RA. Since a significant proportion of ACPAs can be of the IgG4 subclass, the formation of anti–IgG4 hinge antibodies may represent one mechanism of ACPA-mediated inflammation.

Therapeutic monoclonal antibodies are effective drugs for many different diseases. However, the formation of anti-drug antibodies (ADA) against a biological can result in reduced clinical response in some patients. Measurement of ADA in the presence of (high) drug levels is difficult due to drug interference in most assays, including the commonly used antigen binding test (ABT). We recently published a novel method which enables the measurement of complexed antibodies against adalimumab (an anti-TNF antibody) in the presence of drug. Here we use this pH-shift-anti-idiotype ABT (PIA) to measure anti-adalimumab antibodies (AAA) in 99 rheumatoid arthritis (RA) patients treated for up to 3 years with adalimumab. 53 out of 99 RA patients produced AAA. In 50 of these PIA positive patients, AAA could be detected within the first 28 weeks of treatment. Patients in which AAA could be detected in the PIA after 28 weeks of treatment were more prone to declining adalimumab levels (

Eur J Clin Invest 2010; 40 (1): 4–10 Abstract Background Recent studies indicate a role for complement in the pathogenesis of aortic valve disease. However, the role of naturally occurring anti-complement mediators in this context is unknown. In this study, we have analysed this in three different pathological conditions of the aortic valve: degeneration, atherosclerosis and bacterial endocarditis. Materials and methods Human aortic valves were obtained at autopsy (n = 30): 5 control valves, 10 aortic valves with atherosclerotic changes, 10 aortic valves with degenerative changes and 5 degenerative changed aortic valves with bacterial infection. These valves were analysed immunohistochemically for the presence of activated complement (C3d and C5b9) and the complement inhibitors C1-inh and clusterin. Areas of positivity were then quantified. Results C3d, C5b9 and the complement inhibitors C1-inh and clusterin depositions were mainly found in the endothelium and extracellular matrix in aortic valves. All these mediators were already present in control valves, but the area of positivity increased significantly in response to the different diseases, with the highest increase in response to bacterial endocarditis. Interestingly, in all three aortic diseases, the depositions of complement were significantly more widespread than that of their inhibitors. Conclusions Our study indicates that anti-complement mediators (C1-inh and clusterin) are deposited in diseased aortic valves together with activated complement, indicating an existing counter response against complement locally in the valve. However, deposition of activated complement is significantly more widespread than that of its inhibitors, which could explain ongoing inflammation in those diseased aortic valves.

Production of anti drug antibodies (ADA) in adalimumab treated RA patients is associated with reduced serum adalimumab levels and less clinical response. However, most current assays to measure ADA are unable to detect ADA in complex with adalimumab. Thus, ADA is only measured if antibody production exceeds drug levels in the serum, meaning that ADA formation is underestimated. The aim of this study is to develop a method to detect ADA in the presence of drug. A pH-shift-anti-idiotype Antigen binding test (PIA) was used to enable ADA measurement in the presence of adalimumab. ADA-adalimumab complexes were dissociated by acid treatment and addition of excess rabbit anti-idiotype-F(ab) before neutralization. Rabbit anti-idiotype-F(ab) blocks reformation of ADA-drug complexes by competing with patient ADA for adalimumab binding. Released ADA are measured by an antigen binding test (ABT). The PIA enabled detection of ADA in the presence of large excess of adalimumab and was used to measure ADA in 30 adalimumab treated rheumatoid arthritis (RA) patients during the first 28 weeks of treatment. It revealed ADA in 21 out of 30 tested patients, while the ABT detected ADA in only 5 patients. Indicating that an immunogenic reaction towards adalimumab is present in the majority of adalimumab treated patients. (C) 2010 Elsevier B.V. All rights reserved

C1 inhibitor is a potent anti-inflammatory protein as it is the major inhibitor of proteases of the contact and the complement systems. C1-inhibitor administration is an effective therapy in the treatment of patients with hereditary angioedema (HAE) who are genetically deficient in C1 inhibitor. Owing to its ability to modulate the contact and complement systems and the convincing safety profile, plasma-derived C1 inhibitor is an attractive therapeutic protein to treat inflammatory diseases other than HAE. In the present review we give an overview of the biology of C1 inhibitor and its use in HAE. Furthermore, we discuss C1 inhibitor as an experimental therapy in diseases such as sepsis and myocardial infarction.

Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.

Cell-mediated immunity is essential in protection against rickettsial illnesses, but the role of neutrophils in these intracellular vasculotropic infections remains unclear. This study analyzed the plasma levels of nucleosomes, FSAP-activation (nucleosome-releasing factor), and neutrophil activation, as evidenced by neutrophil-elastase (ELA) complexes, in sympatric Lao patients with scrub typhus and murine typhus. In acute scrub typhus elevated nucleosome levels correlated with lower GCS scores, raised respiratory rate, jaundice and impaired liver function, whereas neutrophil activation correlated with fibrinolysis and high IL-8 plasma levels, a recently identified predictor of severe disease and mortality. Nucleosome and ELA complex levels were associated with a 4.8-fold and 4-fold increased risk of developing severe scrub typhus, beyond cut off values of 1,040 U/ml for nucleosomes and 275 U/ml for ELA complexes respectively. In murine typhus, nucleosome levels associated with pro-inflammatory cytokines and the duration of illness, while ELA complexes correlated strongly with inflammation markers, jaundice and increased respiratory rates. This study found strong correlations between circulating nucleosomes and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that nucleosomes could originate from neutrophil extracellular trap (NET) degradation. High circulating plasma nucleosomes and ELA complexes represent independent risk factors for developing severe complications in scrub typhus. As nucleosomes and histones exposed on NETs are highly cytotoxic to endothelial cells and are strongly pro-coagulant, neutrophil-derived nucleosomes could contribute to vascular damage, the pro-coagulant state and exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus., Author Summary Tropical rickettsial illnesses, especially scrub typhus and murine typhus, are increasingly recognized as a leading cause of treatable undifferentiated febrile illness in Asia, but remain severely neglected and under appreciated diseases in many areas. In this study we investigated the relationship of markers of neutrophil activation and cell death with disease severity in patients with acute scrub typhus and murine typhus in Laos. These easily measurable circulating markers were associated with a 4 to 5-fold increased risk of developing severe clinical disease manifestations in scrub typhus and represent independent predictors of severe disease, and possibly death. We also found strong correlations between circulating markers of cell death and neutrophil activation in patients with scrub typhus, but not murine typhus, providing indirect evidence that neutrophil extracellular traps could contribute to the vascular damage and pro-coagulant state leading to exacerbation of disease in scrub typhus, thus indicating a detrimental role of neutrophil activation. The data suggest that increased neutrophil activation relates to disease progression and severe complications, and increased plasma levels of nucleosomes and ELA complexes represent independent risk factors for developing severe scrub typhus.

Antibodies against red blood cells (RBCs) can lead to complement activation resulting in an accelerated clearance via complement receptors in the liver (extravascular hemolysis) or leading to intravascular lysis of RBCs. Alloantibodies (e.g. ABO) or autoantibodies to RBC antigens (as seen in autoimmune hemolytic anemia, AIHA) leading to complement activation are potentially harmful and can be - especially when leading to intravascular lysis - fatal(1). Currently, complement activation due to (auto)-antibodies on RBCs is assessed in vitro by using the Coombs test reflecting complement deposition on RBC or by a nonquantitative hemolytic assay reflecting RBC lysis(1-4). However, to assess the efficacy of complement inhibitors, it is mandatory to have quantitative techniques. Here we describe two such techniques. First, an assay to detect C3 and C4 deposition on red blood cells that is induced by antibodies in patient serum is presented. For this, FACS analysis is used with fluorescently labeled anti-C3 or anti-C4 antibodies. Next, a quantitative hemolytic assay is described. In this assay, complement-mediated hemolysis induced by patient serum is measured making use of spectrophotometric detection of the released hemoglobin. Both of these assays are very reproducible and quantitative, facilitating studies of antibody-induced complement activation.

MBL-deficiency has been associated with an increased frequency and severity of infection, in particular in children and under immunocompromized conditions. In an open uncontrolled safety and pharmacokinetic MBL-substitution study using plasma-derived MBL (pdMBL) in MBL-deficient pediatric oncology patients, we found that despite MBL trough levels above 1.0μg/ml MBL functionality was not efficiently restored upon ex vivo testing. PdMBL showed C4-converting activity by itself, indicating the presence of MASPs. Upon incubation of pdMBL with MBL-deficient sera this C4-converting activity was significantly reduced. Depletion of the MASPs from pdMBL, paradoxically, restored the C4-converting activity. Subsequent depletion or inhibition of C1-inh, the major inhibitor of the lectin pathway, in the recipient serum restored the C4-converting activity as well. Complexes between MBL/MASPs and C1-inh (MMC-complexes) were detected after ex vivo substitution of MBL-deficient serum with pdMBL. These MMC-complexes could also be detected in the sera of the patients included in the MBL-substitution study shortly after pdMBL infusion. Altogether, we concluded that active MBL-MASP complexes in pdMBL directly interact with C1-inh in the recipient, leading to the formation of a multimolecular complex between C1-inh and MBL/MASPs, in contrast to the classical pathway where C1r and C1s are dissociated from C1q by C1-inh. Because of the presence of activated MASPs in the current pdMBL products efficient MBL-mediated host protection cannot be expected because of the neutralizing capacity by C1-inh.

The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity.

Background and objective Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related morbidity and mortality. Specific therapy is lacking. We assessed whether C1-inhibitor attenuates lung injury in a ‘two-hit’ TRALI model. Methods Mice were primed with lipopolysaccharide, subsequently TRALI was induced by MHC-I antibodies. In the intervention group, C1-inhibitor was infused concomitantly. Mice were supported with mechanical ventilation. After 2 h, mice were killed, lungs were removed and bronchoalveolar lavage fluid (BALF) was obtained. Results Injection of MHC-I antibodies induced TRALI, illustrated by an increase in wet-to-dry ratio of the lungs, in BALF protein levels and in lung injury scores. TRALI was further characterized by complement activation, demonstrated by increased BALF levels of C3a and C5a. Administration of C1-inhibitor resulted in increased pulmonary C1-inhibitor levels with high activity. C1-inhibitor reduced pulmonary levels of complement C3a associated with improved lung injury scores. However, levels of pro-inflammatory mediators were unaffected. Conclusion In a murine model of TRALI, C1-inhibitor attenuated pulmonary levels of C3a associated with improved lung injury scores, but with persistent high levels of inflammatory cytokines.

Objectives Millions of patients worldwide are treated with therapeutic monoclonal antibodies. These biological therapeutics can be immunogenic, resulting in anti-drug antibody formation which leads to loss of response. Fully human biological agents, such as the anti-tumour necrosis factor α (anti-TNFα) antibody adalimumab, are considered to be weakly immunogenic, but anti-adalimumab antibodies (AAA) were recently detected in more than half of treated patients with rheumatoid arthritis (RA) within 28 weeks of treatment. A study was undertaken to determine the mechanism by which AAA lead to loss of response. Methods The specificity of the repertoire of AAA was investigated in a cohort of 50 AAA-positive RA patients. Inhibition experiments using TNFα and patient-derived anti-adalimumab monoclonal antibodies were performed. Results The antibody response against adalimumab is highly restricted: Fab fragments of a single monoclonal antibody specific for the idiotype of adalimumab inhibited 98.65% (25th–75th percentiles: 98.25–99.90) of the total anti-adalimumab reactivity in serum from 50 AAA-positive patients. The anti-adalimumab response was confined to the TNFα binding region of adalimumab, thereby neutralising its therapeutic efficacy. In line with this restricted specificity, small immune complexes were found in the circulation of AAA-forming patients. Conclusions The humoral immune response against adalimumab is highly restricted and limited to the idiotype of the therapeutic antibody. All antibodies result in functional neutralisation of the drug, thereby providing a mechanism by which AAA formation leads to clinical non-response.

PURPOSE: Brain inflammation occurs during epileptogenesis and may contribute to the development and progression of temporal lobe epilepsy. Recently, several studies have indicated that seizures may also increase specific blood plasma cytokine levels in animal models as well as in human patients with epilepsy, suggesting that peripheral inflammation may serve as a biomarker for epilepsy. Moreover, studies in epilepsy animal models have shown that peripheral inflammation may play either a pathogenic or neuroprotective role. METHODS: We evaluated the inflammatory response in blood plasma after electrically induced status epilepticus (SE) in a rat model for temporal lobe epilepsy. We measured blood plasma levels of the inflammation markers interleukin 1beta (IL-1beta), interleukin 6 (IL-6), by enzyme-linked immunosorbent assays (ELISAs) and C-reactive protein (CRP) by immunoturbidimetry, at 1 day after SE (acute period), at 1 week (during the latent period), and at 2 months after SE, which is the chronic epileptic phase when spontaneous seizures occur. Plasma levels were also measured during pilocarpine-induced SE. These were compared with plasma levels after lipopolysaccharide injection, which causes sepsis. KEY FINDINGS: Although sepsis induced a huge surge in IL-1beta and IL-6 levels, we did not detect a change in IL-1beta, IL-6, or CRP plasma levels at any time point after electrically induced SE compared to control animals. SE induced by pilocarpine produced a rise in IL-6 and CRP but not IL-1beta levels. SIGNIFICANCE: These findings suggest that plasma levels of these inflammatory proteins cannot be used as biomarkers for temporal lobe epileptogenesis.

Allergen–IgE complexes are more efficiently internalized and presented by B cells than allergens alone. It has been suggested that IgG Abs induced by immunotherapy inhibit these processes. Food-allergic patients have high allergen-specific IgG levels. However, the role of these Abs in complex formation and binding to B cells is unknown. To investigate this, we incubated sera of peanut- or cow’s milk–allergic patients with their major allergens to form complexes and added them to EBV-transformed or peripheral blood B cells (PBBCs). Samples of birch pollen-allergic patients were used as control. Complex binding to B cells in presence or absence of blocking Abs to CD23, CD32, complement receptor 1 (CR1, CD35), and/or CR2 (CD21) was determined by flow cytometry. Furthermore, intact and IgG-depleted sera were compared. These experiments showed that allergen–Ab complexes formed in birch pollen, as well as food allergy, contained IgE, IgG1, and IgG4 Abs and bound to B cells. Binding of these complexes to EBV-transformed B cells was completely mediated by CD23, whereas binding to PBBCs was dependent on both CD23 and CR2. This reflected differential receptor expression. Upon IgG depletion, allergen–Ab complexes bound to PBBCs exclusively via CD23. These data indicated that IgG Abs are involved in complex formation. The presence of IgG in allergen–IgE complexes results in binding to B cells via CR2 in addition to CD23. The binding to both CR2 and CD23 may affect Ag processing and presentation, and (may) thereby influence the allergic response.

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The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.

Summary. Background: Factor VII-activating protease (FSAP) is a serine protease that circulates in plasma in its inactive single-chain form and can be activated upon contact with dead cells. When activated by apoptotic cells, FSAP leads to the release of nucleosomes. The serpins C1-inhibitor and α2-antiplasmin are reported to be the major inhibitors of FSAP. However, regulation of FSAP activity by Kunitz-type inhibitors is not well studied. Objectives: To compare the inhibition of FSAP activity and FSAP-induced nucleosome release from apoptotic cells by tissue factor pathway inhibitor (TFPI) with that of C1-inhibitor and α2-antiplasmin. Methods: Apoptotic cells were incubated with plasma or FSAP in presence of the inhibitor, and nucleosome release was analyzed with flow cytometry. Monoclonal antibodies against TFPI and altered forms of TFPI were used to investigate which domains of TFPI contribute to FSAP inhibition. Results and Conclusions: We show that TFPI abrogates FSAP activity and nucleosome release from apoptotic cells. TFPI is a much more efficient inhibitor than C1-inhibitor or α2-antiplasmin. The active site of K2 is required for inhibition of FSAP. A direct binding interaction between FSAP and the C-terminal domain of TFPI is also required for efficient inhibition. Inhibition of FSAP-induced nucleosome release by recombinant TFPI might, in part, explain the anti-inflammatory effects of recombinant TFPI infusion observed in animal and human sepsis.

In a previous study, the authors found persistent presence of acute inflammation markers such as C-reactive protein and complement factors locally in burn wounds. This persistence of acute inflammation may not only delay local burn wound healing but also have a systemic effect, for instance on the heart. Here, the effects of C1 esterase inhibitor (C1inh), an inhibitor of complement activation, on burn wound progression and the heart were analyzed in rats. Dorsal full-thickness burn wounds (2 x 4 cm) were induced on female Wistar rats (n = 14). The rats were divided into two groups (n = 7): a control group (just burns) and a C1inh group. C1inh was administered daily intravenously for 14 days. The burn wound, healthy skin from the hind leg (internal control), and the heart were then fixed in formalin. Tissues were analyzed for granulation tissue formation, reepithelialization, amount and type of infiltrating inflammatory cells (granulocytes and macrophages), and inflammatory markers (complement factors C3 and C4). C1inh treatment significantly reduced the amount of granulation tissue and significantly increased reepithelialization. C1inh also significantly reduced macrophage infiltration. Burns induced infiltration of macrophages into the ventricles of the heart and remarkably also into the atria of the heart. This effect could be counteracted by C1inh. These data show that systemic treatment with C1inh acts at different levels resulting in improved healing locally in burn wounds and systemically reduced inflammation in the heart. Therefore, C1inh might be a possible therapeutic intervention for burn wound patients. (J Burn Care Res 2012;33:544-551)

A substantial part of rheumatoid arthritis (RA) patients is chronically treated with adalimumab. Some of these patients produce antibodies against adalimumab, which correlate with lower serum drug levels and reduced clinical response. Long term exposure to antigens may result in antigen specific IgG4 production as was demonstrated in studies on prolonged exposure to antigens such as different allergens, Factor VIII and IFN-beta. Here, we investigate whether long term treatment of RA patients with the therapeutic monoclonal antibody adalimumab leads to the production of specific IgG4 antibodies. We developed radio immunoassays to detect total IgG or IgG4 against adalimumab and applied these in a cohort of 271 consecutive RA patients during 3 years of adalimumab treatment. In 32 % of the 271 patients antibodies against adalimumab were detectable. IgG4 antibodies were detected in 29 % of the patients. The proportion IgG4 of total IgG against adalimumab varies widely between patients, and IgG4 was found to contribute significantly to the anti drug antibody (ADA) response in some patients. In the immune response against adalimumab in adalimumab-treated RA patients a considerable part of the ADA is IgG4. Although IgG4 is often considered to be harmless due to its lack of effector function, neutralization of adalimumab by IgG4 antibodies will lead to a reduced clinical response

Rheumatoid factors are antibodies directed against IgG that may confound immunogenicity testing for therapeutic monoclonal antibodies. We developed antigen-binding assays to monitor anti-drug-antibody (ADA) responses against infliximab and adalimumab using F(ab')2 fragments of the drug. This avoids possible detection of rheumatoid factor activity. During development of these assays, a number of sera from patients before treatment as well as several healthy control sera were tested positive. None of these sera contained antibodies specific for the therapeutic mAb. Instead, they were found to contain anti-hinge antibodies. We demonstrate that this aspecific antibody binding can be inhibited by adding F(ab')2 of intravenous immunoglobulin (IVIG), which consists of pooled polyclonal IgG derived from plasma. Using this protocol, anti-infliximab antibodies can be measured specifically without interference by anti-hinge antibodies. (C) 2011 Elsevier B.V. All rights reserved

Salivary agglutinin (SAG), also known as gp-340 and Deleted in Malignant Brain Tumours 1, is a glycoprotein that is present in tears, lung fluid and mucosal surfaces along the gastrointestinal tract. It is encoded by the Deleted in Malignant Brain Tumours 1 gene, a member of the Scavenger Receptor Cysteine Rich group B protein superfamily. SAG aggregates bacteria thus promoting their clearance from the oral cavity and activates the complement system. Complement proteins may enter the oral cavity in case of serum leakage, which occurs after mucosal damage. The purpose of this study was to investigate the mode of complement activation. We showed a dose-dependent C4 deposition on SAG-coated microplates showing that either the classical or lectin pathway of complement was activated. Antibodies against mannose binding lectin inhibited C4 deposition and SAG induced no C4 deposition in MBL deficient sera showing SAG activated complement through the MBL pathway. Periodate treatment of SAG abolished MBL pathway activation consistent with an involvement of SAG glycans in complement activation. This provides the first evidence for a role of SAG in complement activation through the MBL pathway and suggests a potential role of SAG as a complement activating factor at the mucosal epithelia. (C) 2011 Elsevier Ltd. All rights reserved.

The presence of anti-drug antibodies (ADA) in adalimumab-treated patients is associated with reduced serum adalimumab levels and a lower clinical response. Currently, there is no standard for measurement of anti-drug antibodies and many factors influence the results. Consequently, the incidence of ADA as reported in different studies varies considerably. Here we investigated the differential effect of drug interference in two common types of assays used to measure anti-adalimumab: an antigen binding test (ABT) and a more often-used bridging elisa. We measured ADA to adalimumab in a cohort of 216 rheumatoid arthritis patients treated with adalimumab for 28 weeks. Only 15 samples (7%) were positive in the bridging elisa, compared to 29 (13%) in the ABT, despite the fact that the bridging elisa was the most sensitive assay. Furthermore, in an ABT specific for IgG4, 48 samples (22%) were found positive. The bridging elisa was found to detect only the bivalent form of (drug-specific) IgG4, resulting in an underestimation of ADA levels. However, the predominant reason for the different outcomes of these assays was a differential susceptibility to drug interference. In particular, the bridging elisa only detected ADA in the absence of detectable amounts of circulating adalimumab and is therefore not suited for measurement of ADA in complex with the drug. In summary, we showed that a bridging elisa is susceptible to drug interference and typically measures ADA only in absence of detectable drug levels. (C) 2011 Elsevier B.V. All rights reserved

It has been reported that complement is activated on the surface of activated platelets, despite the presence of multiple regulators of complement activation. To reinvestigate the mechanisms by which activated platelets bind to complement components, the presence of complement proteins on the surfaces of nonactivated and thrombin receptor-activating peptide-activated platelets was analyzed by flow cytometry and Western blot analyses. C1q, C4, C3, and C9 were found to bind to thrombin receptor-activating peptide-activated platelets in lepirudin-anticoagulated platelet-rich plasma (PRP) and whole blood. However, inhibiting complement activation at the C1q or C3 level did not block the binding of C3 to activated platelets. Diluting PRP and chelating divalent cations also had no effect, further indicating that the deposition of complement components was independent of complement activation. Furthermore, washed, activated platelets bound added C1q and C3 to the same extent as platelets in PRP. The use of mAbs against different forms of C3 demonstrated that the bound C3 consisted of C3(H2O). Furthermore, exogenously added soluble complement receptor 1 was shown to bind to this form of platelet-bound C3. These observations indicate that there is no complement activation on the surface of platelets under physiological conditions. This situation is in direct contrast to a number of pathological conditions in which regulators of complement activation are lacking and thrombocytopenia and thrombotic disease are the ultimate result. However, the generation of C3(H2O) represents nonproteolytic activation of C3 and after factor I cleavage may act as a ligand for receptor binding.

BACKGROUND: The plasma protein C1-inhibitor (C1-inh), belongs to the serpin superfamily and is the major inhibitor of the proteases of the complement and contact phase pathways. Hereditary or acquired deficiency of functional C1-inh results in angioedema episodes in affected individuals due to uncontrolled contact pathway activation and therapeutic C1-inh products are effective treatment for these patients. Therapeutic C1-inh products have also been shown to attenuate neutrophil activation and infiltration in various inflammatory conditions. This 'novel' anti-inflammatory effect of C1-inh is attributed to its non-serpin N-terminal domain. This domain is thought to express the tetrasaccharide, sialyl Lewisx (SLeX), through which C1-inh can interact with selectins on inflamed endothelium and prevent neutrophil rolling. However, C1-inh products contain small but significant amounts of co-purified proteins, the major one being the glycoprotein α1- antichymotrypsin (ACT), which is also an anti-inflammatory serpin. The potential influence of the glycans of ACT on SLeX - selectin interactions is not clear. METHOD: We investigated the presence of SLeX -like epitopes on C1-inh and ACT from commercially available therapeutic C1-inh preparations using western blotting and mass-spectrometry. The influence of the products and separated C1-inh and ACT on SLeX -selectin interaction was investigated in an a model system where SLeX -beads were rolled on immobilized E-selectin molecules. RESULT: We do not find any evidence of SLeX on C1-inh using either western blotting with anti-SLeX antibodies or by mass spectrometric analysis of C1-inh N- glycans. C1-inh products show modest but significant interference in SLeX -selectin interaction but surprisingly this is not observed for 'pure C1-inh' obtained from gel-filtration of the commercial product. On the contrary, ACT, also from the C1-inh product, shows the presence of SLeX -like epitopes, as detected by the antibody HECA-452 on western blot. In addition, at concentrations present in C1-inh products (20 -150 μg ACT/ mg active C1-inh), ACT can interfere with SLeX -selectin interactions, in a sialic acid dependent manner. These concentrations of ACT can be achieved in vivo with a dose of as low as 2000 U of a C1-inh product, suggesting that ACT can contribute to the anti-inflammatory effects observed in studies with C1-inh products. CONCLUSION: We conclude that the 'novel' anti-inflammatory effects of C1-inh are unlikely due to SLeX and can in fact be partly due to ACT. This fresh evidence challenges a long held assumption and paves the way for development of ACT, alone or in combination with C1-inh, as a new anti-inflammatory therapeutic. Disclosures Engel: ViroPharma Inc.: Research Funding. Nunez:ViroPharma Inc.: Research Funding. Roem:ViroPharma Inc.: Research Funding. van Mierlo:ViroPharma Inc.: Research Funding. Wouters:ViroPharma: Research Funding. Zeerleder:ViroPharma: Other: Receives an unrestricted grant from Viropharma.

Background A substantial part of the rheumatoid arthritis (RA) patients is chronically treated with adalimumab. Some of these patients produce antibodies against adalimumab, which correlate with lower serum drug levels and reduced clinical response. Long term exposure to antigens may result in antigen specific IgG4 production as demonstrated in previous studies on prolonged exposure to antigens such as bee venom, factor VIII and IFN-β. Objectives Here we investigate whether long term treatment of RA patients with the therapeutic monoclonal antibody adalimumab leads to the production of specific IgG4 antibodies. Methods We developed radio immunoassays to detect total IgG or IgG4 against adalimumab and applied these in a cohort of 272 RA patients during three years of adalimumab treatment. Results Of the 272 patients 32% developed antibodies against adalimumab. We observed that a fast majority of the patients also produce IgG4 antibodies against adalimumab (29%). The proportion IgG4 of total IgG against adalimumab varies widely between patients (median; 25-75 percentile 38; 21-67%). In some patients the antibody response even seems to be dominated by IgG4. Conclusions The role of IgG4 in the immune response against adalimumab is variable in adalimumab treated RA patients. Although IgG4 is often considered to be harmless, because of its lack of effector function, these patients show low adalimumab levels and reduced clinical response. Disclosure of Interest None Declared

Abstract 716 In autoimmune hemolytic anemia (AIHA) autoantibodies (auto-Ab's) to red blood cells (RBCs) may induce complement activation via the classical pathway of complement resulting in complement deposition on RBCs. RBCs coated with auto-Ab and/or complement (C3b) are removed by extravascular destruction in the spleen and liver. In a minority of cases complement activation proceeds until insertion of the membrane attack complex (MAC) resulting in intravascular hemolysis. The inflammatory response and the anoxia induced by hemolysis significantly contribute to the morbidity and fatality in AIHA. However, the recovery of RBC transfusion in these situations is inadequate since auto-Abs to RBC also react with donor cells leading to rapid destruction of RBC. Activation of classical pathway of complement is critically involved in this process. C1-inhibitor (Cetor) is a plasma-derived inhibitor of the classical pathway of complement with an excellent safety profile. We hypothesized that co-administration of C1-inhibitor concentrate with a RBC transfusion improves recovery of a transfusion by attenuation of the activation of the classical pathway of complement. In order to test this hypothesis we adapted a standard haemolytic assay (CH50). Sera from patients (n=6) with AIHA due to IgM antibodies with reactivity above 30°C induced dose-dependent lysis of bromealin-treated RBC, whereas sera from healthy donors (n=4) did not. Addition of C1-inhibitor concentrate dose-dependently inhibited RBC lysis significantly in this model. Subsequently, by flowcytometry we studied C3 deposition on RBC after incubation with AIHA patient sera or sera from healthy controls. Anti-C5 antibody (eculizumab) was added in order to prevent lysis during analysis. AIHA sera containing eculizumab induced extensive C3 deposition on RBC, which was significantly inhibited by C1-inhibitor concentrate. This inhibition was dose-dependent. Next, we were interested whether these in-vitro effects could also be observed in-vivo. A 65 year old patient suffering from fulminant life threatening hemolysis due to IgM type auto-Abs reactive >30°C was admitted to our department. Despite treatment with anti-CD20, vincristine and high-dose methylprednisolone the anaemia did not improve necessitating transfusion of 3 RBC concentrates. However, 2.5 day after transfusion the starting haemoglobin levels was reached again and hemolysis deteriorated. Since the patient became symptomatic again the next transfusion of 3 RBC concentrates was preceded by infusion of 6000 U of C1-inhibitor. Three additional donations of 4000 U, 2000 U and 1000 U of C1-inhibitor concentrate have been administered 12, 24 and 36 hours after the first dose, respectively. The recovery of the transfusion significantly increased and was accompanied by a decrease in C3 deposition on RBC, as evidenced by an attenuation of the direct antiglobulin test. In conclusion we have demonstrated that in-vitro C1-inhibitor concentrate can efficiently inhibit RBC lysis and C3 deposition on RBC induced by sera from patients with AIHA. Moreover we have shown that this approach is also effecitive in-vivo. Therefore, in AIHA administration of C1-inhibitor concentrate might be life-saving by improving recovery of RBC transfusion by inhibition of both intra- and extravascular destruction of recipient and donor RBCs. Disclosures: Off Label Use: C1-inhibitor-inhibition of complement-induced hemolysis.