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Complement-mediated autoimmune hemolytic anemia (CM-AIHA) is characterized by destruction of red blood cells (RBCs) by autoantibodies that activate the classical complement pathway. These antibodies also reduce transfusion efficacy via lysis of donor RBCs. Since C1-inhibitor (C1-INH) is an endogenous regulator of the classical complement pathway, we hypothesized that peritransfusional C1-INH in patients with severe CM-AIHA reduces complement activation and hemolysis, and thus enhance RBC transfusion efficacy. We conducted a prospective, single center, phase 2 open-label trial (EudraCT2012-003710-13). Patients with confirmed CM-AIHA and indication for the transfusion of two RBC units were eligible for inclusion. Four intravenous C1-INH doses (6000, 3000, 2000 and 1000 U) were given with 12 h intervals around RBC transfusion. Serial blood samples were analyzed for hemolytic activity, RBC opsonization, complement activation and inflammation markers. Ten patients were included in the study. C1-INH administration increased plasma C1-INH antigen and -activity, peaking at 48 h after the first dose, accompanied by a significant reduction of RBC C3d deposition. Hemoglobin levels increased briefly after transfusion but returned to baseline within 48 h. Overall, markers of hemolysis, inflammation and complement activation remained unchanged. Five grade 3 and one grade 4 adverse event occurred, but were considered unrelated to the study medication. In conclusion, peritransfusional C1-INH temporarily reduced complement activation. However, C1-INH failed to halt hemolytic activity in severe transfusion-dependent CM-AIHA. We cannot exclude that posttransfusional hemolytic activity would have been even higher without C1-INH. The potential of complement inhibition on transfusion efficacy in severe CM-AIHA remains to be determined.

Introduction: Plasma exchange therapy (PEX) was standard treatment for thrombotic microangiopathy before eculizumab was available and is still widely applied. However, most PEX patients still ultimately progress to end-stage renal disease (ESRD). It has been suggested that infusion of plasma that contains active complement may induce additional complement activation with subsequent activation of neutrophils and endothelial cells, leading to exacerbation of organ damage and deterioration of renal function. Objective: This observational pilot study examines the effect of hemodialysis, eculizumab and PEX before and after treatment in plasma of aHUS patients on complement-, neutrophil and endothelial cell activation. Methods: Eleven patients were included in this pilot study. Six patients were treated with hemodialysis, 2 patients received regular infusions of eculizumab, and 3 patients were on a regular schedule for PEX. Patients were followed during 3 consecutive treatments. Blood samples were taken before and after patients received their treatment. Results: Complement activation products increased in plasma of patients after PEX, as opposed to patients treated with hemodialysis or eculizumab. Increased levels of complement activation products were detected in omniplasma used for PEX. Additionally, activation of neutrophils and endothelial cells was observed in patients after hemodialysis and PEX, but not in patients receiving eculizumab treatment. Conclusion: In this pilot study we observed that PEX induced complement and neutrophil activation, and that omniplasma contains significant amounts of complement activation products. Additionally, we demonstrate that hemodialysis induces activation of neutrophils and endothelial cells. Complement activation with subsequent neutrophil activation may contribute to the deterioration of organ function and may result in ESRD. Further randomized controlled studies are warranted to investigate the effect of PEX on complement- and neutrophil activation in patients with thrombotic microangiopathy.

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.

Correct glycosylation of proteins is essential for production of therapeutic proteins as glycosylation is important for protein solubility, stability, half-life and immunogenicity. The heavily glycosylated plasma protein C1-inhibitor (C1-INH) is used in treatment of hereditary angioedema attacks. In this study, we used C1-INH as a model protein to propose an approach to develop recombinant glycoproteins with the desired glycosylation. We produced fully functional recombinant C1-INH in Chinese hamster ovary (CHO) cells. In vivo we observed a biphasic clearance, indicating different glycosylation forms. N-glycan analysis with mass spectrometry indeed demonstrated heterogeneous glycosylation for recombinant C1-INH containing terminal galactose and terminal sialic acid. Using a Ricinus Communis Agglutinin I (RCA120) column, we could reduce the relative abundance of terminal galactose and increase the relative abundance of terminal sialic acid. This resulted in a fully active protein with a similar in vivo clearance rate to plasmaderived C1-INH. In summary, we describe the development of a recombinant human glycoprotein using simple screening tools to obtain a product that is similar in function and in vivo clearance rate to its plasma-derived counterpart. The approach used here is of potential use in the development of other therapeutic recombinant human glycoproteins.

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.

The complement system represents the first line of defense that is involved in the clearance of pathogens, dying cells and immune complexes via opsonization, induction of an inflammatory response and the formation of a lytic pore. Red blood cells (RBCs) are very important for the delivery of oxygen to tissues and are continuously in contact with complement proteins in the blood plasma. To prevent complement activation on RBCs, various complement regulatory proteins can be found in plasma and on the cell membrane. RBCs are special cells without a nucleus and having a slightly different make-up of complement regulators than nucleated cells, as membrane cofactor protein (MCP) is not expressed and complement receptor 1 (CR1) is highly expressed. Decreased expression and/or function of complement regulatory proteins may result in unwanted complement activation and accelerated removal of RBCs. This review describes complement regulation on RBCs and the consequences when this regulation is out of balance.

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.

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.

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.

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.

INTRODUCTION: Atrial fibrillation (AF) is a common complication in myocarditis. Atrial inflammation has been suggested to play an important role in the pathophysiology of AF. However, little is known about the occurrence of atrial inflammation in myocarditis patients. Here, we analyzed inflammatory cell numbers in the atria of myocarditis patients without symptomatic AF.METHODS: Cardiac tissue was obtained postmortem from lymphocytic myocarditis patients (n=6), catecholamine-induced myocarditis patients (n=5), and control patients without pathological evidence of heart disease (n=5). Tissue sections of left and right ventricle and left and right atrium were stained for myeloperoxidase (neutrophilic granulocytes), CD45 (lymphocytes), and CD68 (macrophages). These cells were subsequently quantified in atrial and ventricular myocardium and atrial adipose tissue.RESULTS: In lymphocytic myocarditis patients, a significant increase was observed for lymphocytes in the left atrial adipose tissue. In catecholamine-induced myocarditis patients, significant increases were found in the atria for all three inflammatory cell types. Infiltrating inflammatory cell numbers in the atrial myocardium correlated positively with those in the ventricles, especially in catecholamine-induced myocarditis patients.CONCLUSIONS: To a varying extent, atrial myocarditis occurs concurrently with ventricular myocarditis in patients diagnosed with myocarditis of different etiology. This provides a substrate that potentially predisposes myocarditis patients to the development of AF and subsequent complications such as sudden cardiac death and heart failure.

AIMS: Inflammation plays an important role in the pathogenesis of myocardial infarction (MI). Whether MI induces atrial inflammation is unknown however. Here, we analysed atrial inflammation in patients with MI and in rats with experimentally induced MI. The effect of the anti-inflammatory agent C1-esterase inhibitor (C1inh) on atrial inflammation in rats was also analysed.METHODS: In the hearts of patients who died at different time points after MI (total n=24, mean age=60), neutrophils (myeloperoxidase-positive cells), lymphocytes (CD45-positive cells) and macrophages (CD68-positive cells) were quantified in the myocardium of the left and right atria and the infarcted left and non-infarcted right ventricles and compared with control patients (n=5, mean age=59). For the left and right atria, inflammatory cells were also quantified in the atrial adipose tissue. MI was induced in 17 rats, of which 10 were subsequently treated with C1inh for 6 days. Forty-two days post-MI, lymphocytes, macrophages and the endothelial inflammation marker Nε-(carboxymethyl)lysine (CML) were analysed in the myocardium of both the atria and ventricles.RESULTS: In all investigated areas of the human hearts increased lymphocytes and macrophages were observed to a varying extent, especially between 6 h and 5 days following MI. Similarly, in rats MI resulted in an increase of inflammatory cells and CML in the atria. C1inh treatment decreased atrial inflammation.CONCLUSIONS: MI induces atrial inflammation in patients and in rats. C1inh treatment could counteract this MI-induced atrial inflammation in rats.

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

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.

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 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.

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.