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Neisseria meningitidis, the causative agent of meningococcal disease (MD), evades complement-mediated clearance upon infection by ‘hijacking’ the human complement regulator factor H (FH). The FH protein family also comprises the homologous FH-related (FHR) proteins, hypothesized to act as antagonists of FH, and FHR-3 has recently been implicated to play a major role in MD susceptibility. Here, we show that the circulating levels of all FH family proteins, not only FH and FHR-3, are equally decreased during the acute illness. We did neither observe specific consumption of FH or FHR-3 byN. meningitidis, nor of any of the other FH family proteins, suggesting that the globally reduced levels are due to systemic processes including dilution by fluid administration upon admission and vascular leakage. MD severity associated predominantly with a loss of FH rather than FHRs. Additionally, low FH levels associated with renal failure, suggesting insufficient protection of host tissue by the active protection by the FH protein family, which is reminiscent of reduced FH activity in hemolytic uremic syndrome. Retaining higher levels of FH may thus limit tissue injury during MD.

Neisseria meningitidis, the causative agent of meningococcal disease (MD), evades complement-mediated clearance upon infection by ‘hijacking’ the human complement regulator factor H (FH). The FH protein family also comprises the homologous FH-related (FHR) proteins, hypothesized to act as antagonists of FH, and FHR-3 has recently been implicated to play a major role in MD susceptibility. Here, we show that, next to FH and FHR-3, the circulating levels of all FH family proteins are equally decreased during pediatric MD. We did not observe a specific consumption of FH or FHR-3 by N. meningitidis during the first days of infection and the levels recovered over time. MD severity associated predominantly with a loss of FH. Strikingly, loss of FH and FHRs associated strongly with renal failure, suggesting insufficient protection of host tissue by the FH protein family. Retaining higher levels of FH family proteins may thus limit tissue injury during MD.

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 To prevent unwanted complement activation and subsequent damage, complement activation must be tightly regulated on healthy host cells. Dysregulation of the complement system contributes to the pathology of diseases like Paroxysmal Nocturnal Hemoglobinuria and atypical Hemolytic Uremic Syndrome. To investigate complement regulator deficiencies, primary patient cells may be used, but access to patient cells may be limited and cells are heterogeneous between different patients. To inhibit regulator function on healthy host cells, blocking antibodies can be used, though it may be difficult to exclude antibody-mediated effects. To circumvent these issues, we created single and combined complement regulator human knockout cells to be able to in vitro investigate complement activation and regulation on human cells. Methods CRISPR/Cas9 was used to knockout (KO) complement regulatory proteins CD46, CD55 and/or CD59 in human HAP1 cells. Single cell derived cell lines were profiled by Sanger sequencing and flow cytometry. To confirm the lack of complement regulatory function, the cells were exposed to complement in normal human serum and subsequently C3 and C4 deposition on the cell surface were detected by using flow cytometry. Results We created single KO cell lines that completely lacked CD46, CD55 or CD59. We additionally generated double CD46/CD55, CD46/CD59 and CD55/CD59 KOs and triple CD46/CD55/CD59 KOs. Upon classical pathway activation, deletion of CD46 resulted in increased C3 and C4 deposition, while deleting CD55 mainly resulted to increased C3 deposition, confirming their reported function in complement regulation. Upon alternative pathway activation, C3 deposition was only observed on the triple CD46/CD55/CD59 KO cells and not on any of the other cell lines, suggesting that human cells are resistant to spontaneous complement activation and suggesting a role for CD59 in C3 regulation. Conclusions The generation of complement regulator KO cell lines provides a relevant tool for future in vitro investigations of complement activation and regulation on human cells. Furthermore, these cell lines may also be helpful to evaluate therapeutic complement inhibitors and may shed light on novel roles of complement regulatory proteins as we here observed for CD59.

Oncological treatment has been associated with an increased risk of infection, most often related to therapy-induced pancytopenia. However, limited research has been conducted on the effect of oncological therapy on the complement system, being part of the non-cellular innate immune system. This became the rationale for an observational clinical study (C2012) in which we have investigated the prevalence of transient complement defects. Once we had observed such defects, a correlation of the complement defects to specific clinical parameters or to specific therapeutic regimens was investigated. A prominent defect observed in C2012 was the inhibition of the lectin pathway (LP) of complement activation during the treatment of acute lymphoblastic leukemia (ALL), which we could directly associate to the use of asparaginase (ASNase). Ex-vivo experiments confirmed a direct dose-dependent inhibitory effect of ASNase on the LP functionality.

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.

There is an error in the Acknowledgments statement. The correct name for the funder “Spanish Ministerio de Economia y Competitividad” is “Spanish Instituto de Salud Carlos III.” A correction has been made to the Acknowledgments section: “We appreciate the technical assistance of Lorena Risueno, Cesar Velez, and Alex Otero in the management of biological samples of patients and controls. We thank Rosario Madero (IdiPAZ Biostatistics) for statistical help, and Vega Mauleon and Hoi Tong (IdiPAZ UCICEC) for blood sampling of the healthy Spanish volunteers. This study was funded by the Spanish Instituto de Salud Carlos III and the European Program FEDER (grants PI12/00597 and PI16/00723 to PS-C, and grant SAF2015-66287-R to SR), and by the Complement II-CM network (B2017/BMD-3673). RP and TK received funding from the European Union’s seventh Framework program under ECGA no. 279185 (EUCLIDS; www.euclids-project.eu). AL is supported by the Spanish Center for Biomedical Network Research on Rare Diseases (CIBERER).” The authors apologize for this error and state that this does not change the scientific conclusions of the article in any way. The original article has been updated.

Background The complement system is a vital component of the inflammatory response occurring during bacterial meningitis. Blocking the complement system was shown to improve the outcome of experimental pneumococcal meningitis. Complement factor H (FH) is a complement regulatory protein inhibiting alternative pathway activation but is also exploited by the pneumococcus to prevent complement activation on its surface conferring serum resistance. Methods In a nationwide prospective cohort study of 1009 episodes with community-acquired bacterial meningitis, we analyzed whether genetic variations in CFH influenced FH cerebrospinal fluid levels and/or disease severity. Subsequently, we analyzed the role of FH in our pneumococcal meningitis mouse model using FH knock-out (Cfh−/−) mice and wild-type (wt) mice. Finally, we tested whether adjuvant treatment with human FH (hFH) improved outcome in a randomized investigator blinded trial in a pneumococcal meningitis mouse model. Results We found the major allele (G) of single nucleotide polymorphism in CFH (rs6677604) to be associated with low FH cerebrospinal fluid concentration and increased mortality. In patients and mice with bacterial meningitis, FH concentrations were elevated during disease and Cfh−/− mice with pneumococcal meningitis had increased mortality compared to wild-type mice due to C3 depletion. Adjuvant treatment of wild-type mice with purified human FH led to complement inhibition but also increased bacterial outgrowth which resulted in similar disease outcomes. Conclusion Low FH levels contribute to mortality in pneumococcal meningitis but adjuvant treatment with FH at a clinically relevant time point is not beneficial.

BACKGROUND: There is a clinical need for immunosuppressive therapy that can treat myocarditis patients in the presence of an active viral infection. In this study we therefore investigated the effects of colchicine, an immunosuppressive drug which has been used successfully as treatment for pericarditis patients, in a mouse model of coxsackievirus B3(CVB3)-induced myocarditis.METHODS: Four groups of C3H mice were included: control mice (n=8), mice infected with CVB3 (1×10(5) PFU, n=10), mice with colchicine administration (2mg/kg i.p, n=5) and mice with combined CVB3 infection and colchicine administration (n=10). After three days, the heart, pancreas and spleen were harvested and evaluated using (immuno)histochemical analysis and CVB3 qPCR.RESULTS: Mice were terminated at day 3 post-virus infection as colchicine treatment rapidly resulted in severe illness and mortality in CVB3-infected mice. Colchicine significantly decreased the number of macrophages in the heart in CVB3-infected mice (pCONCLUSIONS: Colchicine treatment in CVB3-induced myocarditis has a detrimental effect as it causes complete destruction of the exocrine pancreas and enhances viral load in both heart and pancreas.

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

Background Plasmodium falciparum may evade complement-mediated host defense by hijacking complement Factor H (FH), a negative regulator of the alternative complement pathway. Plasma levels of FH vary between individuals and may therefore influence malaria susceptibility and severity. Methods We measured convalescent FH plasma levels in 149 Gambian children who had recovered from uncomplicated or severe P. falciparum malaria and in 173 healthy control children. We compared FH plasma levels between children with malaria and healthy controls, and between children with severe (n = 82) and uncomplicated malaria (n = 67). We determined associations between FH plasma levels and laboratory features of severity and used multivariate analyses to examine associations with FH when accounting for other determinants of severity. Results FH plasma levels differed significantly between controls, uncomplicated malaria cases, and severe malaria cases (mean [95% confidence interval], 257 [250 to 264], 288 [268 to 309], and 328 [313 to 344] µg/mL, respectively; analysis of variance P < .0001). FH plasma levels correlated with severity biomarkers, including lactate, parasitemia, and parasite density, but did not correlate with levels of PfHRP2, which represent the total body parasite load. Associations with severity and lactate remained significant when adjusting for age and parasite load. Conclusions Natural variation in FH plasma levels is associated with malaria susceptibility and severity. A prospective study will be needed to strengthen evidence for causation, but our findings suggest that interfering with FH binding by P. falciparum might be useful for malaria prevention or treatment.

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.

Improved methods are needed to identify host mechanisms which directly protect against human infectious diseases in order to develop better vaccines and therapeutics1,2. Pathogen load determines the outcome of many infections3, and is a consequence of pathogen multiplication rate, duration of the infection, and inhibition or killing of pathogen by the host (resistance). If these determinants of pathogen load could be quantified then their mechanistic correlates might be determined. In humans the timing of infection is rarely known and treatment cannot usually be withheld to monitor serial changes in pathogen load and host response. Here we present an approach to overcome this and identify potential mechanisms of resistance which control parasite load inPlasmodium falciparummalaria. Using a mathematical model of longitudinal infection dynamics for orientation, we made individualized estimates of parasite multiplication and growth inhibition in Gambian children at presentation with acute malaria and used whole blood RNA-sequencing to identify their correlates. We identified novel roles for secreted proteases cathepsin G and matrix metallopeptidase 9 (MMP9) as direct effector molecules which inhibitP. falciparumgrowth. Cathepsin G acts on the erythrocyte membrane, cleaving surface receptors required for parasite invasion, whilst MMP9 acts on the parasite. In contrast, the type 1 interferon response and expression ofCXCL10(IFN-γ-inducible protein of 10 kDa, IP-10) were detrimental to control of parasite growth. Natural variation in iron status and plasma levels of complement factor H were determinants of parasite multiplication rate. Our findings demonstrate the importance of accounting for the dynamic interaction between host and pathogen when seeking to identify correlates of protection, and reveal novel mechanisms controlling parasite growth in humans. This approach could be extended to identify additional mechanistic correlates of natural- and vaccine-induced immunity to malaria and other infections.

Recent research has elucidated circulating levels of almost all factor H-related (FHR) proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH), fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs) that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 +/- 1.46 mu g/mL) were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

C1q is the initiation molecule of the classical pathway of the complement system and is produced by macrophages and immature dendritic cells. As mast cells share the same myeloid progenitor cells, we have studied whether also mast cells can produce and secrete C1q. Mast cells were generated in vitro from CD34+ progenitor cells from buffy coats or cord blood. Fully differentiated mast cells were shown by both RNA sequencing and qPCR to express C1QA, C1QB and C1QC. C1q produced by mast cells has a similar molecular make-up as serum C1q. Reconstituting C1q depleted serum with mast cell supernatant in haemolytic assays, indicated that C1q secreted by mast cells is functionally active. The level of C1q in supernatants produced under basal conditions was considerably enhanced upon stimulation with LPS, dexamethasone in combination with IFN-gamma or via Fc epsilon RI triggering. Mast cells in human tissues stained positive for C1q in both healthy and in inflamed tissue. Moreover, mast cells in healthy and diseased skin appear to be the predominant C1q positive cells. Together, our data reveal that mast cells are able to produce and secrete functional active C1q and indicate mast cells as a local source of C1q in human tissue. (C) 2016 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license

Recent research has elucidated circulating levels of almost all factor H-related (FHR) proteins. Some of these proteins are hypothesized to act as antagonists of the important complement regulator factor H (FH), fine-tuning complement regulation on human surfaces. For the CFHR4 splice variants FHR-4A and FHR-4B, the individual circulating levels are unknown, with only total levels being described. Specific reagents for FHR-4A or FHR-4B are lacking due to the fact that the unique domains in FHR-4A show high sequence similarity with FHR-4B, making it challenging to distinguish them. We developed an assay that specifically measures FHR-4A using novel, well-characterized monoclonal antibodies (mAbs) that target unique domains in FHR-4A only. Using various FHR-4A/FHR-4B-specific mAbs, no FHR-4B was identified in any of the serum samples tested. The results demonstrate that FHR-4A is the dominant splice variant of CFHR4 in the circulation, while casting doubt on the presence of FHR-4B. FHR-4A levels (avg. 2.55 ± 1.46 µg/mL) were within the range of most of the previously reported levels for all other FHRs. FHR-4A was found to be highly variable among the population, suggesting a strong genetic regulation. These results shed light on the physiological relevance of the previously proposed role of FHR-4A and FHR-4B as antagonists of FH in the circulation.

Dysregulation of the complement alternative pathway (AP) is a major pathogenic mechanism in atypical hemolytic-uremic syndrome (aHUS). Genetic or acquired defects in factor H (FH), the main AP regulator, are major aHUS drivers that associate with a poor prognosis. FH activity has been suggested to be downregulated by homologous FH-related (FHR) proteins, including FHR-3 and FHR-1. Hence, their relative levels in plasma could be disease-relevant. The genes coding for FH, FHR-3, and FHR-1 (; CFH, CFHR3; , and; CFHR1; , respectively) are polymorphic and located adjacent to each other on human chromosome 1q31.3. We have previously shown that haplotype; CFH(H3)-CFHR3*B-CFHR1*B; associates with aHUS and reduced FH levels. In this study, we used a specific enzyme-linked immunosorbent assay to quantify FHR-3 in plasma samples from controls and patients with aHUS genotyped for the three known; CFHR3; alleles (; CFHR3*A, CFHR3*B; , and; CFHR3*Del; ). In the 218 patients carrying at least one copy of; CFHR3; , significant differences between; CFHR3; genotype groups were found, with; CFHR3*A/Del; patients having the lowest FHR-3 concentration (0.684-1.032 µg/mL),; CFHR3*B/Del; and; CFHR3*A/A; patients presenting intermediate levels (1.437-2.201 µg/mL), and; CFHR3*A/B; and; CFHR3*B/B; patients showing the highest concentration (2.330-4.056 µg/mL) (; p

BACKGROUND Human immunodeficiency virus (HIV)-induced complement activation may play a role in chronic immune activation in patients with HIV infection and influence the complement system during acute illness. We determined the impact of HIV infection on the complement system in patients with asymptomatic HIV infection and HIV-infected patients with sepsis or malaria. METHODS We performed a prospective observational study of 268 subjects with or without HIV infection who were asymptomatic, were septic, or had malaria. We measured complement activation products (C3bc and C4bc) and native complement proteins (C3 and C4). levels of mannose-binding lectin and C1q-C4 were measured to examine activation of the lectin and classical pathways, respectively. RESULTS Asymptomatic HIV infection was associated with increased C4 activation, especially in patients with high HIV loads, and was accompanied by elevated C1q-C4 levels. Similarly, sepsis and malaria resulted in increased C4 activation and elevated C1q-C4 concentrations. HIV coinfection enhanced C4 activation and consumption in patients with sepsis; this effect was not detected in patients with malaria. Mannose-binding lectin deficiency (defined as a mannose-binding lectin level of

Human cytomegalovirus (CMV) induces the formation of effector CD8(+) T cells that are maintained for decades during the latent stage of infection. Effector CD8(+) T cells appear quiescent, but maintain constitutive cytolytic capacity and can immediately produce inflammatory cytokines such as IFN-γ after stimulation. It is unclear how effector CD8(+) T cells can be constitutively maintained in a terminal stage of effector differentiation in the absence of overt viral replication. We have recently described the zinc finger protein Homologue of Blimp-1 in T cells (Hobit) in murine NKT cells. Here, we show that human Hobit was uniformly expressed in effector-type CD8(+) T cells, but not in naive or in most memory CD8(+) T cells. Human CMV-specific but not influenza-specific CD8(+) T cells expressed high levels of Hobit. Consistent with the high homology between the DNA-binding Zinc Finger domains of Hobit and Blimp-1, Hobit displayed transcriptional activity at Blimp-1 target sites. Expression of Hobit strongly correlated with T-bet and IFN-γ expression within the CD8(+) T cell population. Furthermore, Hobit was both necessary and sufficient for the production of IFN-γ. These data implicate Hobit as a novel transcriptional regulator in quiescent human effector-type CD8(+) T cells that regulates their immediate effector functions.

Asthma patients show evidence of a procoagulant state in their airways, accompanied by an impaired function of the anticoagulant protein C system. We aimed to study the effect of recombinant human activated protein C (rhAPC) in allergic asthma patients.We conducted a randomised, double-blind, placebo-controlled, proof-of-concept study in house dust mite (HDM) allergic asthma patients. Patients were randomised to receive intravenous rhAPC (24 µg·kg−1·h−1; n=12) or placebo (n=12) for 11 h. 4 h after the start of infusion, a first bronchoscopy was performed to challenge one lung segment with saline (control) and a contralateral segment with a combination of HDM extract and lipopolysaccharide (HDM+LPS), thereby mimicking environmental house dust exposure. A second bronchoscopy was conducted 8 h after intrabronchial challenge to obtain bronchoalveolar lavage fluid (BALF).rhAPC did not influence HDM+LPS induced procoagulant changes in the lung. In contrast, rhAPC reduced BALF leukocyte counts by 43% relative to placebo, caused by an inhibitory effect on neutrophil influx (64% reduction), while leaving eosinophil influx unaltered. rhAPC also reduced neutrophil degranulation products in the airways.Intravenous rhAPC attenuates HDM+LPS-induced neutrophil migration and protein release in allergic asthma patients by an effect that does not rely on coagulation inhibition.

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.

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.

C1 esterase inhibitor (C1-INH) can inhibit multiple pathways (complement, contact-kinin, coagulation, and fibrinolysis) that are all implicated in the pathophysiology of asthma. We explored the effect of human plasma-derived C1-INH on allergic lung inflammation in a house dust mite (HDM) induced asthma mouse model by daily administration of C1-INH (15 U) during the challenge phase. NaCl and HDM exposed mice had comparable plasma C1-INH levels, while bronchoalveolar lavage fluid (BALF) levels were increased in HDM exposed mice coinciding with slightly reduced activation of complement (C5a). C1-INH treatment reduced Th2 response and enhanced HDM-specific IgG1. Influx of eosinophils in BALF or lung, pulmonary damage, mucus production, procoagulant response or plasma leakage in BALF was similar in both groups. In conclusion, C1-INH dampens Th2 responses during HDM induced allergic lung inflammation.

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.

The lectin pathway (LP) of complement has a protective function against invading pathogens. Recent studies have also shown that the LP plays an important role in ischemia/reperfusion (I/R)-injury. MBL-associated serine protease (MASP)-2 appears to be crucial in this process. The serpin C1-inhibitor is the major inhibitor of MASP-2. In addition, aprotinin, a Kunitz-type inhibitor, was shown to inhibit MASP-2 activity in vitro. In this study we investigated whether the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI) is also able to inhibit MASP-2. Ex vivo LP was induced and detected by C4-deposition on mannan-coated plates. The MASP-2 activity was measured in a fluid-phase chromogenic assay. rTFPI in the absence or presence of specific monoclonal antibodies was used to investigate which TFPI-domains contribute to MASP-2 inhibition. Here, we identify TFPI as a novel selective inhibitor of MASP-2, without affecting MASP-1 or the classical pathway proteases C1s and C1r. Kunitz-2 domain of TFPI is required for the inhibition of MASP-2. Considering the role of MASP-2 in complement-mediated I/R-injury, the inhibition of this protease by TFPI could be an interesting therapeutic approach to limit the tissue damage in conditions such as cerebral stroke, myocardial infarction or solid organ transplantation.

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.

Immunogenicity is a major issue of concern for monoclonal antibodies used in human diseases and is by default mainly determined in non-human primates (NHP), as target molecules are considered most similar in NHP compared to human. In this manuscript the predictive value of immunogenicity testing in minipigs for human safety is evaluated, as the immune system of the pig is functionally similar to that in other mammalian species. Adalimumab and infliximab (both monoclonal antibodies blocking TNFα) were used as model substances. Female Gottingen minipigs (4/group) were treated every other week with low (0.1mg/kg), mid (1.0mg/kg), or high dose (5mg/kg) adalimumab or 5mg/kg infliximab subcutaneous (SC) over a period of 8 weeks. After first and last dosing, pharmacokinetic analysis was performed. Anti-drug antibodies (ADAs) were measured on several time points. Furthermore, hematology, clinical chemistry, body weight, clinical signs, and histopathology of several organs were evaluated. No signs of toxicity of the treatments were observed in the limited organs and tissues collected. Eleven out of 12 minipigs treated with adalimumab elicited a detectable ADA response. Induction of ADA was correlated with decreased plasma levels of adalimumab. Infliximab clearance was comparable after first and last dose. Therefore, the presence of ADA directed to infliximab was considered highly unlikely. It was concluded that the minipig and NHP showed comparable suitability for immunogenicity prediction in humans. More studies with other biopharmaceutical products are needed to strengthen the status of the minipig as an alternative model for immunotoxicity testing including immunogenicity. © 2014 Informa Healthcare USA, Inc. Chemicals/CAS: adalimumab, 331731-18-1; infliximab, 170277-31-3 Tradenames: humira, abbott biotechnology, Germany; remicade, Centocor, Netherlands Manufacturers: abbott biotechnology, Germany; Centocor, Netherlands

MBL-deficiency is a commonly occurring deficiency of the innate immune system, affecting a substantial part of the population and has been extensively studied. MBL appears to function as a disease modifier. The role of MBL in different conditions is context-dependent. Many clinical studies show conflicting results, which can be partially explained by different definitions of MBL-deficiency, including phenotype- and genotype-based approaches. In this review we give an overview of literature of MBL, its role in different pathologies, diseases and patient populations. We review MBL replacement studies, and discuss the potential of MBL substitution therapy. We finally suggest that new MBL substitution trials should be conducted within a predefined patient population. MBL-deficiency should be based on serum levels and confirmed by genotyping. (C) 2014 Elsevier Ltd. All rights reserved

Activation of complement may cause severe tissue damage in antibody-mediated allograft rejection and other antibody-mediated clinical conditions; therefore, novel potent complement inhibitors are needed. Previously, we described binding of the inhibitory receptor LAIR-1 and its soluble family member LAIR-2 to collagen. Here, we investigated binding of LAIR-1 and LAIR-2 to the complement proteins C1q and MBL, which both have collagen-like domains, and evaluated the effect of this binding on complement function. We demonstrate specific binding of recombinant LAIR proteins to both C1q and MBL. Surface plasmon resonance experiments showed that LAIR-2-Fc protein bound C1q and MBL with the highest affinity compared to LAIR-2-HIS. We, therefore, hypothesized that LAIR-2-Fc is a potent complement inhibitor. Indeed, LAIR-2-Fc inhibited C4 fixation to IgG or mannan, reduced activation of C4 by aggregated IgG in plasma and inhibited iC3b deposition on cells. Finally, LAIR-2-Fc inhibited complement-mediated lysis of cells sensitized with anti-HLA antibodies in an ex vivo model for antibody-mediated transplant rejection. Thus, LAIR-2-Fc is an effective novel complement inhibitor for the treatment and prevention of antibody-mediated allograft rejection and antibody-mediated clinical conditions. (C) 2013 S. Karger AG, Basel

Contains fulltext : 177971.pdf (Publisher’s version ) (Open Access) PURPOSE: A clear link between several variants in genes involved in the complement system and chronic central serous chorioretinopathy (CSC) has been described. In age-related macular degeneration, a disease that shows clinical features that overlap with CSC, both genetic risk factors and systemic activation of the complement system have previously been found. In this case-control study, we assessed whether there is evidence of either systemic activation or inhibition of the complement system in patients with chronic CSC. METHODS: A prospective case-control study of 76 typical chronic CSC patients and 29 controls without ophthalmological history was conducted. Complement activity assays (classical, alternative, and mannose-binding lectin pathway), complement factors 3, 4, 4A, 4B, B, D, H, I, and P, activation products C3d, C5a, and sC5b-C9, and the C3d/C3 ratio were analysed in either serum or plasma. A correction for possible effects of gender, age, body mass index, and smoking status was performed. RESULTS: In this study, none of the tested variables, including regulation and activation products, proved to be significantly different between the groups. Moreover, no associations with either CSC disease activity or possible CSC related steroid use were observed. CONCLUSION: Despite the available literature regarding a possible relationship between chronic CSC and variants in genes involved in the complement system, we did not find evidence of an association of chronic CSC with either systemic complement activation or inhibition.

BACKGROUND: Adipose-derived stromal cells (ASCs) are a promising new therapeutic option for patients with acute myocardial infarction (AMI). Previously, we found that ASCs coupled to antibody-targeted microbubbles (StemBells [StBs]) improved cardiac function when administered intravenously 7 days post-AMI in rats. In this study, we compared the efficacy of intravenous StB administration at different administration time points following AMI in rats.METHODS: AMI, followed by reperfusion, was induced in four groups of male Wistar rats, which subsequently received an intravenous 1 × 10(6) StB bolus 1 day post-AMI (StB1; n = 8), 7 days post-AMI (StB7; n = 9), at both time points (StB1+7; n = 7) or neither (Control; n = 7). The effect onrdiac function was determined using echocardiography prior to AMI, 7 days post-AMI and 42 days post-AMI. The effect on infarct size and macrophages in the infarct core were determined (immuno)histochemically 42 days post-AMI.RESULTS: At 42 days post-AMI, all three StB groups had a significantly improved fractional shortening compared with the control group. Between the StB-treated groups, the effects did not differ significantly at 42 days post-AMI. At 7 days post-AMI, the StB1 group had a significantly improved fractional shortening compared with the control and StB7 groups. No significant changes in infarct size or macrophage numbers were found compared with the control group for any StB group.CONCLUSIONS: StB administration resulted in long-term improvement of cardiac function, independent of the time point of administration. When administered at 1 day post-AMI, this improvement was already evident at 7 days post-AMI.

Activation of antigen-presenting dendritic cells (DCs) and the complement system are essential early events in the immune defense against invading pathogens. Recently, we and others demonstrated immunological crosstalk between signaling from receptors recognizing complement activation products and PAMPs on DCs. This affects DC effector function, as demonstrated by the finding that C5a prevents induction of pro-inflammatory cytokines by toll-like receptor (TLR) ligands in human monocyte-derived DCs (moDCs). Here, we demonstrate that this regulatory crosstalk is specifically important in 6-sulfo LacNAc dendritic cells (slanDCs), the most pro-inflammatory DC subset found in human. C5aR and TLR signaling show profound interference in the ERK/p38/CREB1 signaling pathways. C5aR signaling accelerates TLR-induced CREB1 phosphorylation both in moDC and slanDC. This is key in the regulatory effect of C5a on pro-inflammatory DC maturation by mediating induction of IL-10, which subsequently inhibits pro-inflammatory cytokine production via negative feedback signaling. Importantly, the regulatory effect of C5a affects T-cell immunity by decreasing Th1 and cytotoxic CD8 T-cell responses. The finding that the pro-inflammatory effector function of slanDC can be down modulated by activation products of the complement system highlights the existence of intricate regulatory interactions between various arms of the immune system. Intensive immune monitoring of patients suffering from complement-mediated diseases or patients receiving complement modulating compounds can give more inside in the contribution of complement receptor and TLR crosstalk in APCs in disease.

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 (

Introduction To date, oncology patients are more dependent on non-cellular host defense against pathogens due to intensive (chemo)therapy-related bone marrow suppression. Since data on complement functionality in oncology patients are limited, we aimed to investigate the innate complement function in relation to the type of malignancy and therapy in a longitudinal cohort of patients. Methods A large single-center, prospective non-intervention study was conducted, in which blood samples were taken from patients before, during, and after treatment with chemotherapy and/or subsequent admittance for (febrile) neutropenia. Results/findings Analysis of 48 patients showed a high percentage of defects in complement activity of the alternative pathway (19.1%), the classical pathway (4.3%), or both (42.6%). Post hoc analysis of six different treatment protocols with more than three patients each showed distinct effects of specific therapies. Whereas patients treated according to the Ewing, EpSSG-rhabdomyosarcoma, or SIOP CNS germ cell tumor protocol showed no defects, patients treated according to the ALL-11 (leukemia), the EURAMOS I (osteosarcoma), or the ACNS (medulloblastoma) protocols showed an almost universal reduction in complement function. Although we could not explain the reduced complement functionality under all conditions, a strong effect was observed following high-dose methotrexate or ifosfamide. Conclusion Acquired complement defects were commonly observed in more than 50% of oncology patients, some of which associated with certain chemotherapeutic drugs. Additional studies are needed to determine the clinical and therapeutic context of complement defects and their possible effect on treatment outcome or the increased risk of infection.

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

Complement factor C4 exists as two main isotypes, C4A and C4B, with different functional properties and encoded by two separate genes. In addition, C4A and C4B genes can occur in multiple copies and may carry a retroviral HERV-K(C4) insertion in intron 9. To study association of C4 polymorphism with disease, accurate genotyping and phenotyping is important. However, current techniques are very laborious and not suitable to study large patient groups. Therefore, we aimed to develop novel assays for C4 geno- and phenotyping, to make high throughput possible. To study C4 gene copy number variation, a novel Multiplex Ligation-dependent Probe Amplification (MLPA) assay was set up with three synthetic probe parts. C4A and C4B protein levels were measured by isotype-specific ELISA's. The relationship between C4 genotype with C4A and C4B serum concentrations was examined in 104 healthy tab workers and 66 children with meningococcal disease. As expected, a strong positive correlation was found between C4A and C4B gene copy number and serum levels of total C4, C4A and C4B. In the healthy controls, 95.3% of C4A genes and 53.7% of C4B genes carried the HERV-K(C4) insertion. Presence of HERV-K(C4) resulted in less C4B protein expression, while there was no effect on total C4 levels. In the meningitis patients, no increased incidence of hetero- or homozygous deficiency of either C4A or C4B was found. In conclusion, the combination of MLPA and ELISA is very suitable to study the geno- and phenotype of complement C4 in large patient groups. (C) 2008 Elsevier Ltd. All rights reserved