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The lepirudin-based human whole blood model is a well-established ex vivo system to characterize inflammatory responses. However, the contribution of individual cell populations to cytokine release has not been investigated. Thus, we modified the model by selectively removing leukocyte subpopulations to elucidate their contribution to the inflammatory response. Lepirudin-anticoagulated whole blood was depleted from monocytes or granulocytes using StraightFrom Whole Blood MicroBeads. Reconstituted blood was incubated with Escherichia coli(108/mL) for 2 hours at 37 °C. CD11b, CD62P, and CD63 were detected by flow cytometry. Complement (C3bc, sC5b-9) and platelet activation (platelet factor 4, NAP-2) were measured by enzyme-linked immunosorbent assay. Cytokines were quantified by multiplex assay. A significant (P< 0.05) specific depletion of the monocyte (mean = 86%; 95% confidence interval = 71%–92%) and granulocyte (mean = 97%; 95% confidence interval = 96%–98%) population was obtained. Background activation induced by the depletion protocol was negligible for complement (C3bc and sC5b-9), leukocytes (CD11b), and platelets (NAP-2). Upon Escherichia coliincubation, release of 10 of the 24 cytokines was solely dependent on monocytes (interleukin [IL]-1β, IL-2, IL-4, IL-5, IL-17A, interferon-γ, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein-1α, and fibroblast growth factor–basic), whereas 8 were dependent on both monocytes and granulocytes (IL-1ra, IL-6, IL-8, IL-9, IL-10, macrophage inflammatory protein-1β, tumor necrosis factor, and eotaxin). Six cytokines were not monocyte or granulocyte dependent, of which platelet-derived growth factor and RANTES were mainly platelet dependent. We document an effective model for selective depletion of leukocyte subpopulations from whole blood, without causing background activation, allowing in-depth cellular characterization. The results are in accordance with monocytes playing a major role in cytokine release and expand our knowledge of the significant role of granulocytes in the response to E. coli.By selective removal of leukocyte subtypes from human blood, we were able to use a holistic approach to identify cells responsible for the cytokine release induced by Escherichia coliincubated in the whole blood model.

The urinary tract is constantly exposed to microorganisms. Host defense mechanisms in protection from microbial colonization and development of urinary tract infections require better understanding to control kidney infection. Here we report that the lectin collectin 11 (CL-11), particularly kidney produced, has a pivotal role in host defense against uropathogen infection. CL-11 was found in mouse urine under normal and pathological conditions. Mice with global gene ablation of Colec11had increased susceptibility to and severity of kidney and to an extent, bladder infection. Mice with kidney-specific Colec11ablation exhibited a similar disease phenotype to that observed in global Colec11deficient mice, indicating the importance of kidney produced CL-11 for protection against kidney and bladder infection. Conversely, intravesical or systemic administration of recombinant CL-11 reduced susceptibility to and severity of kidney and bladder infection. Mechanism analysis revealed that CL-11 can mediate several key innate defense mechanisms (agglutination, anti- adhesion, opsonophagocytosis), and limit local inflammatory responses to pathogens. Furthermore, CL-11-mediated innate defense mechanisms can act on clinically relevant microorganisms including multiple antibiotic resistant strains. CL-11 was detectable in eight of 24 urine samples from patients with urinary tract infections but not detectable in urine samples from ten healthy individuals. Thus, our findings demonstrate that CL-11 is a key factor of host defense mechanisms in kidney and bladder infection with therapeutic potential for human application.

Despite the success of mRNA-based vaccines against infectious diseases (including COVID-19), safety concerns have been raised relating to the lipid nanoparticles (LNPs) used to deliver the mRNA cargo. Antibodies against the polyethylene glycol (PEG) coating on these non-viral vectors are present in the general population and can in some instances induce allergic reactions. Furthermore, treatment with PEGylated therapeutics may increase the plasma concentration of such anti-PEG antibodies. The widespread use of PEGylated nanoparticles for mRNA vaccines concerns researchers and clinicians about a potential rise in future cases of allergic reactions against mRNA vaccines and cross-reactions with other PEGylated therapeutics. To determine if vaccination with Comirnaty increased the plasma concentration of antibodies against LNPs, we investigated the blood plasma concentration of anti-LNP antibodies in healthy individuals before and after vaccination with the mRNA-based COVID-19 vaccine Comirnaty (BNT162b2). Blood samples were acquired from 21 healthy adults before vaccination, 3–4 weeks after the first vaccination dose but before the second dose, and 2–6 months after the second (booster) dose. The blood plasma concentration of antibodies recognizing the LNPs was analyzed using a microscopy-based assay capable of measuring antibody-binding to individual authentic LNPs. No significant increase in anti-LNP antibodies was observed after two doses of Comirnaty. The LNPs used for intramuscular delivery of mRNA in the vaccine against COVID-19, Comirnaty, do, therefore, not seem to induce the generation of anti-vector antibodies.

Mannose-binding lectin-associated serine protease 2 (MASP-2) is the main activator of the lectin complement pathway and has been suggested to be involved in the pathophysiology of coronavirus disease 2019 (COVID-19). To study a possible association between MASP-2 and COVID-19, we aimed at developing a sensitive and reliable MASP-2 ELISA. From an array of novel mouse-monoclonal antibodies using recombinant MASP-2 as antigen, two clones were selected to create a sandwich ELISA. Plasma samples were obtained from 216 healthy controls, 347 convalescent COVID-19 patients, and 147 prospectively followed COVID-19 patients. The assay was specific towards MASP-2 and did not recognize the truncated MASP2splice variant MAP-2 (MAp19). The limit of quantification was shown to be 0.1 ng/mL. MASP-2 concentration was found to be stable after multiple freeze-thaw cycles. In healthy controls, the mean MASP-2 concentration was 524 ng/mL (95% CI: 496.5–551.6). No significant difference was found in the MASP-2 concentrations between COVID-19 convalescent samples and controls. However, a significant increase was observed in prospectively followed COVID-19 patients (mean: 834 ng/mL [95% CI: 765.3–902.7, p< 0.0001]). In these patients, MASP-2 concentration correlated significantly with the concentrations of the terminal complement complex (ρ = 0.3596, p< 0.0001), with the lectin pathway pattern recognition molecules ficolin-2 (ρ = 0.2906, p= 0.0004) and ficolin-3 (ρ = 0.3952, p< 0.0001) and with C-reactive protein (ρ = 0.3292, p= 0.0002). Overall, we developed a specific quantitative MASP-2 sandwich ELISA. MASP-2 correlated with complement activation and inflammatory markers in COVID-19 patients, underscoring a possible role of MASP-2 in COVID-19 pathophysiology.

Introduction:The complement system anaphylatoxin C5a is a critical player in inflammation. By binding to complement C5a receptor 1 (C5aR1/CD88), C5a regulates many cellular functions, mainly as a potent pro-inflammatory inducer. We describe the generation and selection of a potent antagonistic C5aR1 mouse monoclonal antibody (mAb). Methods:Initial C5aR1 hybridoma clone selection was performed with a cell-binding study in human whole blood. In-house C5aR1 mAb assessment for C5aR1 inhibition was done via the iLite®C5a assay. C5aR1 mAb specificity was investigated on C5aR1his- and C5aR2his-expressing Flp-In™-CHO cells. Physiological C5aR1 inhibition was assessed via a C5a-driven calcium flux assay and stimulation assay based on isolated polymorphonuclear leukocytes (PMNs) and a whole blood model stimulated with Escherichia coli. Results:The supernatant of hybridoma clones targeting the N-terminal section of C5aR1 displayed efficient binding to C5aR1 in whole blood, which was confirmed for purified mAbs. The C5aR1 mAb 18-41-6 was selected following the assay of in-house C5aR1 mAbs via the iLite®C5a assay. The mAb 18-41-6 was specific for C5aR1. Full-size and/or F(ab’)2preparations of mAb 18-41-6 were found to efficiently abrogate C5a-induced calcium flux in neutrophils and to significantly reduce the upregulation of the activation markers CD11b (neutrophils, monocytes) and CD66b (neutrophils). Conclusion:Our results demonstrate that mAb 18-41-6 is a valuable tool for investigating the C5a-C5aR1 axis and a potential therapeutic candidate for inflammatory disease treatment.

The hyperinflammatory burden is immense in necrotizing soft-tissue infection (NSTI). The complement system is a key during the innate immune response and may be a promising target to reduce the inflammatory response, potentially improving the clinical outcome. However, complement activation and its association to disease severity and survival remain unknown in NSTI. Therefore, we prospectively enrolled patients with NSTI and sampled blood at admission and once daily for the following 3 days. Plasma C4c, C4d, C3bc, and C3dg and the terminal complement complex (TCC) were evaluated using ELISA techniques. In total, 242 patients were included with a median age of 62 years, with a 60% male predominance. All-cause 30-day mortality was 17% (95% confidence interval [CI] 13–23) with a follow-up of >98%. C4c and C3dg were negatively correlated with Simplified Acute Physiology Score II (Rho−0.22, p< 0.001 and Rho−0.17, p= 0.01). Patients with septic shock (n= 114, 47%) had higher levels of baseline TCC than those in non-shock patients (18 vs. 14, p< 0.001). TCC correlated with the Sequential Organ Failure Assessment (SOFA) score (Rho0.19, p= 0.004). In multivariate Cox regression analysis (adjusted for age, sex, comorbidity, and SOFA score), high baseline C4d (>20 ng/mL) and the combination of high C4d and TCC (>31 arbitrary units/mL) were associated with increased 30-day mortality (hazard ratio [HR] 3.26, 95% CI 1.56–6.81 and HR 5.12, 95% CI 2.15–12.23, respectively). High levels of both C4d and TCC demonstrated a negative predictive value of 0.87. In conclusion, we found that in patients with NSTI, complement activation correlated with the severity of the disease. High baseline C4d and combination of high C4d and TCC are associated with increased 30-day mortality. Low baseline C4d or TCC indicates a higher probability of survival.

The humoral arm of innate immunity includes diverse molecules with antibody-like functions, some of which serve as disease severity biomarkers in coronavirus disease 2019 (COVID-19). The present study was designed to conduct a systematic investigation of the interaction of human humoral fluid-phase pattern recognition molecules (PRMs) with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Of 12 PRMs tested, the long pentraxin 3 (PTX3) and mannose-binding lectin (MBL) bound the viral nucleocapsid and spike proteins, respectively. MBL bound trimeric spike protein, including that of variants of concern (VoC), in a glycan-dependent manner and inhibited SARS-CoV-2 in three in vitro models. Moreover, after binding to spike protein, MBL activated the lectin pathway of complement activation. Based on retention of glycosylation sites and modeling, MBL was predicted to recognize the Omicron VoC. Genetic polymorphisms at the MBL2locus were associated with disease severity. These results suggest that selected humoral fluid-phase PRMs can play an important role in resistance to, and pathogenesis of, COVID-19, a finding with translational implications.

The course of COVID-19 is unpredictable, ranging from asymptomatic to respiratory failure and death. Prognostic biomarkers are urgently needed. We hypothesized that long pentraxin PTX3 could be a valuable plasma biomarker due to its essential role in inflammatory processes. In a prospective hospitalized COVID-19 derivation cohort (n= 126) during the spring of 2020, we measured PTX3 within 4 days of admission. The predictive value of mechanical ventilation (MV) and 30-day mortality compared with clinical parameters and other markers of inflammation were assessed by logistic regression analysis and expressed as odds ratio (OR) with 95% confidence interval (CI). Analyses were repeated in a prospective validation cohort (n= 112) of hospitalized patients with COVID-19 treated with remdesivir and dexamethasone. Thirty-day mortality in the derivation cohort was 26.2%. In patients who died, the median PTX3 concentration upon admission was 19.5 ng/mL (IQR: 12.5–33.3) versus 6.6 ng/mL (IQR 2.9–12.3) (p< 0.0001) for survivors. After adjustment for covariates, the odds of 30-day mortality increased two-fold for each doubling of PTX3 (OR 2.03 [95% CI: 1.23–3.34], p= 0.006), which was also observed in the validation cohort (OR 1.70 [95% CI: 1.09–2.67], p= 0.02). Similarly, PTX3 levels were associated with MV. After adjustment for covariates, OR of MV was 2.34 (95% CI: 1.33–4.12, p= 0.003) in the derivation cohort and 1.64 (95% CI: 1.03–2.62, p= 0.04) in the validation cohort. PTX3 appears to be a useful clinical biomarker to predict 30-day respiratory failure and mortality risk in COVID-19 patients treated with and without remdesivir and dexamethasone.

Serological assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are needed to support clinical diagnosis and epidemiological investigations. Recently, assays for large-scale detection of total antibodies (Ab), immunoglobulin G (IgG), and IgM against SARS-CoV-2 antigens have been developed, but there are limited data on the diagnostic accuracy of these assays.

Activation of the complement system is mediated by the interaction between pathogens and pattern recognition molecules (PRMs); mannose-binding lectin (MBL), ficolins, and collectin-10/-11 from the lectin pathway and C1q from the classical pathway. Lectin pathway activation specifically depends on proteases named MBL-associated serine proteases (MASPs) that are found in complexes with PRMs. In this study, we hypothesize that MASPs can recognize selected pathogens independently of PRMs. Using different clinical strains of opportunistic fungi, we have observed that MASPs directly recognize certain fungal pathogens in a way that can facilitate complement activation. Among these were Aspergillus fumigatus– a dangerous pathogen, especially for immunocompromised patients. In flow cytometry and fluorescence microscopy, we found that MASP-1 and -3 bound to all A. fumigatusgrowth stages (conidia, germ tubes, and hyphae), whereas rMASP-2 and the nonproteolytic rMAP-1 did not. Bound rMASPs could recruit rMBL and rficolin-3 to A. fumigatusconidia in a nonclassical manner and activate complement via rMASP-2. In experiments using recombinant and purified components, rMASP-1 increased the neutrophilic phagocytosis of conidia. In serum where known complement activation pathways were blocked, phagocytosis could be mediated by rMASP-3. We have encountered an unknown pathway for complement activation and found that MASP-1 and MASP-3 have dual functions as enzymes and as PRMs.

The complement system was discovered at the end of the 19th century as a heat-labile plasma component that “complemented” the antibodies in killing microbes, hence the name “complement.” Complement is also part of the innate immune system, protecting the host by recognition of pathogen-associated molecular patterns. However, complement is multifunctional far beyond infectious defense. It contributes to organ development, such as sculpting neuron synapses, promoting tissue regeneration and repair, and rapidly engaging and synergizing with a number of processes, including hemostasis leading to thromboinflammation. Complement is a double-edged sword. Although it usually protects the host, it may cause tissue damage when dysregulated or overactivated, such as in the systemic inflammatory reaction seen in trauma and sepsis and severe coronavirus disease 2019 (COVID-19). Damage-associated molecular patterns generated during ischemia-reperfusion injuries (myocardial infarction, stroke, and transplant dysfunction) and in chronic neurologic and rheumatic disease activate complement, thereby increasing damaging inflammation. Despite the long list of diseases with potential for ameliorating complement modulation, only a few rare diseases are approved for clinical treatment targeting complement. Those currently being efficiently treated include paroxysmal nocturnal hemoglobinuria, atypical hemolytic-uremic syndrome, myasthenia gravis, and neuromyelitis optica spectrum disorders. Rare diseases, unfortunately, preclude robust clinical trials. The increasing evidence for complement as a pathogenetic driver in many more common diseases suggests an opportunity for future complement therapy, which, however, requires robust clinical trials; one ongoing example is COVID-19 disease. The current review aims to discuss complement in disease pathogenesis and discuss future pharmacological strategies to treat these diseases with complement-targeted therapies.Significance StatementThe complement system is the host’s defense friend by protecting it from invading pathogens, promoting tissue repair, and maintaining homeostasis. Complement is a double-edged sword, since when dysregulated or overactivated it becomes the host’s enemy, leading to tissue damage, organ failure, and, in worst case, death. A number of acute and chronic diseases are candidates for pharmacological treatment to avoid complement-dependent damage, ranging from the well established treatment for rare diseases to possible future treatment of large patient groups like the pandemic coronavirus disease 2019.

Animal and observational studies have suggested a pathophysiological role for complement in venous thromboembolism (VTE), but the initiating mechanisms are unknown. Mannose‐binding lectin (MBL) bound to altered host cells leads to activation of the lectin complement pathway, and both high and low MBL levels have been implicated in the pathophysiology of cardiovascular disease.

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Aspergillus fumigatusis an opportunistic fungal pathogen that causes severe invasive infections in immunocompromised patients. Innate immunity plays a major role in protection against A. fumigatus.The ficolins are a family of soluble pattern recognition receptors that are capable of activating the lectin pathway of complement. Previous in vitro studies reported that ficolins bind to A. fumigatus, but their part in host defense against fungal infections in vivo is unknown. In this study, we used ficolin-deficient mice to investigate the role of ficolins during lung infection with A. fumigatus. Ficolin knockout mice showed significantly higher fungal loads in the lungs 24 h postinfection compared to wild-type mice. The delayed clearance of A. fumigatusin ficolin knockout mice could not be attributed to a compromised recruitment of inflammatory cells. However, it was revealed that ficolin knockout mice exhibited a decreased production of proinflammatory cytokines in the lungs compared to wild-type mice following A. fumigatus infection. The impaired clearance and cytokine production in ficolin knockout mice was independent of complement, as shown by equivalent levels of A. fumigatus-mediated complement activation in ficolin knockout mice and wild-type mice. In conclusion, this study demonstrates that ficolins are important in initial innate host defense against A. fumigatusinfections in vivo.

Background:Mannose-binding lectin (MBL) and ficolins are pattern recognition molecules (PRMs) that play an important role during infection through activation of the lectin complement pathway. We assessed whether plasma PRM levels were associated with mortality in patients with necrotizing soft tissue infection (NSTI). Methods:We conducted a prospective, observational study over 25 months involving 135 NSTI patients with a maximum follow-up of 2.7 years. Blood samples were taken upon admission. Non-infected patients served as controls. Results:PRM levels were significantly lower compared with controls. A baseline Ficolin-2 level below the median was associated with mortality at the end of follow-up (p = 0.007). No significant association was found for MBL, Ficolin-1 and Ficolin-3. A Ficolin-2 level below the median had a negative predictive value of 0.94 for 28-day mortality, and a level below the optimal cut-off was independently associated with 28-day mortality when adjusted for age, sex and chronicity [hazard ratio 6.27 (95% confidence interval 2.28-17.21), p < 0.0001], also when Simplified Acute Physiology Score II was included in the analysis [hazard ratio 3.16 (95% confidence interval 1.03-9.73), p = 0.045]. Conclusions:All PRMs were significantly lower in patients with NSTI than in controls. Only baseline Ficolin-2 was associated with short- and long-term mortality. A high baseline Ficolin-2 level indicated a 94% chance of surviving the first 28 days after admission.

Complement activation induced by latex, polyvinylchloride, and silicone tubings for extracorporeal circulation was studied in 2 in vitrosituations: Fresh whole human blood mixed with priming solution was circulated through 00 cm tubing lengths for 1 hour, and small tubing pieces were incubated in fresh human serum for 7 hours. Activation of C3 and of the terminal pathway (C5–C9) was assessed in enzyme immuno assays. In both situations complement activation was significantly higher (p<0.05) than in controls. During incubation, C3 activation started later with silicone, but increased to similar levels as with polyvinylchloride after 7 hours. Latex was a significantly more potent C3 activator at 7 hours than the other materials (p<0.05), whereas polyvinylchloride induced most terminal pathway activation (p<0.01). When blood was pumped through intact tubing, complement activation was similar with all materials. The differences found during incubation were apparently overshadowed by other factors in the dynamic situation. The study thus gives no evidence for preferring any of the tested tubing materials to reduce complement activation during in vivoextracorporeal circulation, even if the tubing does contribute to overall activation.

To assess the involvement of ficolin-3, the main initiator of the lectin complement pathway (LCP), in subarachnoid hemorrhage (SAH) pathology and outcome.

AbstractStaphylococcus aureusis an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first lines of defense against bacterial pathogens, and S. aureusexpresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureusare potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureusmutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureusappear to be important for pathogen-mediated evasion of the human complement system.© 2013 S. Karger AG, Basel

AbstractCollectins and ficolins are important in the clearance of endogenous and exogenous danger materials. A new human collectin-11 was recently identified in low concentration in serum in complex with mannose-binding lectin (MBL)/ficolin-associated serine proteases. Collectin-11 binds to carbohydrate residues present on various microorganisms. Thus, we hypothesized that collectin-11 could be a novel initiation molecule in the lectin pathway of complement. We can show that collectin-11 associates with all the known MBL-associated serine proteases (MASP-1, MASP-2 and MASP-3) as well as the lectin complement pathway regulator MAP-1. Furthermore, we found that complex formation between recombinant collectin-11 and recombinant MASP-2 on Candida albicansleads to deposition of C4b. Native collectin-11 in serum mediated complement activation and deposition of C4b and C3b, and formation of the terminal complement complex on C. albicans. Moreover, spiking collectin-11-depleted serum, which did not mediate complement activation, with recombinant collectin-11 restored the complement activation capability. These results define collectin-11 as the fifth recognition molecule in the lectin complement pathway in addition to MBL, ficolin-1, ficolin-2 and ficolin-3.Copyright © 2012 S. Karger AG, Basel

Coagulation disorders and reperfusion of ischemic myocardium are major causes of morbidity and mortality. Lectin pathway initiation complexes are composed of multimolecular carbohydrate recognition subcomponents and 3 lectin pathway–specific serine proteases. We have recently shown that the lectin pathway–specific carbohydrate recognition subcomponent mannose-binding lectin plays an essential role in the pathophysiology of thrombosis and ischemiareperfusion injury. Thus, we hypothesized that the endogenous mannose-binding lectin (MBL)ficolin-associated protein-1 (MAP-1) that inhibits complement activation in vitro also could be an in vivo regulator by attenuating myocardial schemareperfusion injury and thrombogenesis when used at pharmacological doses in wild-type mice.

OBJECTIVE: To examine whether IgG glycosylation changes and MBL2 genotypes are associated with systemic inflammation and joint destruction in rheumatoid arthritis (RA). METHODS: IgG N-glycan content was determined from serum in 118 patients with RA by high-throughput glycan analysis using normal-phase high-pressure liquid chromatography. MBL2 extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) were determined. Systemic inflammation was assessed by serum levels of C-reactive protein (CRP), interleukin 6 (IL-6), and tumor necrosis factor-α (TNF-α). Joint destruction was assessed by total Sharp score (TSS) and alloplastic surgery records. RESULTS: IgG hypogalactosylation was significantly correlated to IL-6 (Spearman’s rho = 0.32, p < 0.001), CRP (Spearman’s rho = 0.31, p < 0.001), TSS (Spearman’s rho = 0.25, p = 0.01), and alloplastic replacement of joints (Spearman’s rho = 0.18, p = 0.05). In multivariate analysis including age, CRP, anticitrullinated protein antibodies (ACPA), and other confounders, IgG hypogalactosylation was significantly associated with TSS (p = 0.014) and alloplastic joint replacement (OR 76.5, p = 0.041) in patients homozygous for the high expression MBL2 genotype YA/YA, but not in carriers of lower expression genotypes. CONCLUSION: Decreased galactosylation of IgG correlated to markers of inflammation, i.e., IL-6 and CRP. Only in patients homozygous for high expression of the MBL2 genotype YA/YA was IgG hypogalactosylation associated with markers of joint destruction. Our results suggest that inflammation-associated decreased galactosylation of IgG combined with high expression MBL2 genotypes are involved in the pathophysiology of RA.

ABSTRACTIn a cohort study of children <4 years of age in Greenland, mannose-binding lectin (MBL2) genotypes and Epstein-Barr virus (EBV) antibody levels were determined. EBV seropositivity was significantly lower and time to seroconversion increased in MBL-insufficient compared with MBL-sufficient children, indicating that MBL may be involved in primary EBV infection in infancy.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have excess cardiovascular morbidity and mortality due to accelerated atherosclerosis that cannot be attributed to traditional cardiovascular risk factors alone. Variant alleles of the mannose-binding lectin gene (MBL2) causing low serum concentrations of functional mannose-binding lectin (MBL) are associated with SLE and development of atherosclerosis. Recent studies show that these variant alleles are associated with increased risk of arterial thrombosis and cardiovascular disease in patients with SLE. Intima-media thickness of the common carotid artery (ccIMT) is a validated noninvasive anatomic measure of subclinical atherosclerosis. In a cross-sectional study we examined the relation among ccIMT, MBL2 genotypes, and serum concentrations of MBL. METHODS: The MBL2 extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) and serum concentrations of MBL were determined in 41 outpatients with SLE. ccIMT was measured by means of ultrasonography. Traditional and nontraditional cardiovascular risk modifiers were assessed and controlled for. RESULTS: Using nonparametric Mann-Whitney tests we found a significant difference in ccIMT between low-expressing (XA/XA+YA/YO+XA/YO+YO/YO) and high-expressing (YA/YA+YA/XA) MBL2 genotypes (p = 0.034). The difference in ccIMT remained significant in multivariable analysis adjusting for traditional and nontraditional cardiovascular risk modifiers (p = 0.049). ccIMT was negatively correlated to serum concentrations of MBL (Spearman rho = –0.33, p = 0.037). This relation also remained significant in multivariable analysis (p = 0.042). CONCLUSION: In this group of SLE patients, MBL2 low-expressing genotypes and low serum levels of MBL were correlated with ccIMT, independent of the effects of traditional and nontraditional cardiovascular risk modifiers.

Molecular mechanism behind Ficolin‐1 association with leukocytes. Three Ficolins have been identified in humans: Ficolin‐1 (M‐Ficolin), Ficolin‐2 (L‐Ficolin), and Ficolin‐3 (H‐Ficolin). Ficolin‐1 is the least‐described of the Ficolins and is expressed by monocytes, granulocytes, and in the lungs. Ficolin‐1 is found circulating at low concentrations in serum but is regarded primarily as a secretory molecule that exerts its function locally in inflamed tissues. Ficolin‐1 has been reported on the surface of monocytes and granulocytes and was suggested originally to function as a phagocytic receptor. However, the molecule does not contain any obvious transmembrane domain, and no binding partners have been identified. To gain further insight in the physiological role of Ficolin‐1, we sought to identify the molecular mechanism responsible for the membrane association of Ficolin‐1 to monocytes and granulocytes. We demonstrate that expression of Ficolin‐1 on the cell surface is restricted to monocytes and granulocytes. Ficolin‐1 is tethered to the cell surface of these cells through its fibrinogen‐like domain, and the ligand involved in the binding of Ficolin‐1 is shown to be sialic acid. Moreover, rFicolin‐1 bound activated but not resting T lymphocytes. Together, these results demonstrate a novel self‐recognition mechanism of leukocytes mediated by the fibrinogen‐like domain of Ficolin‐1.

OBJECTIVE: Patients with rheumatoid arthritis (RA) have increased overall and cardiovascular mortality. Mannose-binding lectin (MBL) may play differentiated roles in the pathogenesis of RA. We had observed that high serum levels of MBL increased the risk of ischemic heart disease in patients with RA. In this followup study we describe the mortality in a cohort of 229 Danish patients with RA. We examine if previously reported factors and MBL influence the risk of overall death and death due to cardiovascular disease. METHODS: Known predictors of RA mortality were assessed. MBL extended genotypes (YA/YA, YA/XA, XA/XA, YA/YO, XA/YO, YO/YO) were determined; MBL serum concentrations were measured. The vital status and causes of death were assessed in a prospective study. RESULTS: The median followup was 10.3 years. The overall risk of death was 4% per year. Comparing mortality in the RA cohort with mortality in an age- and sex-matched cohort based on the general Danish population, we found significantly increased overall mortality [standardized mortality ratio (SMR) 1.5, 95% CI 1.2–1.9, and cardiovascular mortality (SMR 1.7, 95% CI 1.3–2.6)]. In multivariate analysis, significant predictors of overall death were extraarticular manifestations, positive rheumatoid factor, increased C-reactive protein (CRP), poor nutritional state, and serum MBL. Predictors of cardiovascular death were Health Assessment Questionnaire score, increased CRP, poor nutritional state, and the high-producing MBL genotype YA/YA. CONCLUSION: Both overall and cardiovascular mortality were increased in Danish patients with RA. In our cohort, states of high MBL production and several previously reported factors contributed significantly to this increased risk of overall death and cardiovascular death.

Ficolin‐1 is present in gelatinase granules and also in a previously unknown highly mobilizable subset of granules; once released, ficolin‐1 binds to the neutrophil surface. Ficolins are soluble molecules that bind carbohydrate present on the surface of microorganisms and function as recognition molecules in the lectin complement pathway. Three ficolins have been identified in humans: ficolin‐1, ficolin‐2, and ficolin‐3. Ficolin‐1 is synthesized in monocytes and type II alveolar epithelial cells. Ficolin‐1 has been shown to be present in secretory granules of human neutrophils, but it is not known which subset of the neutrophils’ secretory granules harbors ficolin‐1. To determine the exact subcellular localization of ficolin‐1 in neutrophils, recombinant ficolin‐1 was expressed in Chinese hamster ovary cells and used for generation of polyclonal antibodies. This allowed detection of ficolin‐1 in subcellular fractions of human neutrophils by ELISA, by Western blotting, and by immunohistochemistry. Real‐time PCR examination of normal human bone marrow showed FCN1 gene expression largely in myelocytes, metamyelocytes, and band cells with a profile quite similar to that of gelatinase. In accordance with this, biosynthesis studies of neutrophils precursor cells showed that ficolin‐1 was primarily synthesized in myelocytes, metamyelocytes, and band cells. Immunohistochemistry and subcellular fractionation demonstrated that ficolin‐1 is primarily localized in gelatinase granules but also in highly exocytosable gelatinase‐poor granules, not described previously. Ficolin‐1 is released from neutrophil granules by stimulation with fMLP or PMA, and the majority becomes associated with the surface membrane of the cells and can be detected by flow cytometry. Our studies show that neutrophils are a major source of ficolin‐1, which can be readily exocytosed by stimulation.

AbstractFicolins constitute a family of proteins whose biological role has been an enigma for many years. Over the past few years it has become evident that ficolins are part of the innate immune system and function as recognition molecules in the complement system. The 3 human ficolins, ficolin-1 (M-ficolin), ficolin-2 (L-ficolin) and ficolin-3 (H-ficolin or Hakata antigen) are encoded by the FCN1, FCN2and FCN3genes, respectively. Phylogenetic studies suggest that ficolins are of ancient origin. Ficolin-3 seems to be the most ancient molecule, from a phylogenetic perspective. Searches in databases and phylogenetic tree analysis demonstrate that the ficolin precursor has gone through an expansion involving independent duplication events in the different branches of the evolutionary tree. Of particular interest is the prediction that ficolin-1 appears to be present as an ortholog molecule. All human FCNgenes are polymorphic. The FCN2gene encoding ficolin-2, contains polymorphisms that affect ligand binding, while differences in the serum levels are associated with promoter polymorphisms. Recently, a frame-shift variation in the FCN3gene was described, leading to ficolin-3 deficiency and defective complement activation. This FCN3variation was also shown to be associated with immunodeficiency. This survey summarizes the current phylogenetic and inter-individual molecular understanding of the FCNgenes.Copyright © 2009 S. Karger AG, Basel

OBJECTIVE: To study the association between polymorphisms in the mannose-binding lectin gene (MBL2) and disease activity, physical disability, and joint erosions in patients with newly diagnosed rheumatoid arthritis (RA). METHODS: Patients with early RA (n = 158) not previously treated with disease modifying antirheumatic drugs, participating in a treatment trial (CIMESTRA study) were examined at inclusion for MBL2 pooled structural genotypes (O/O, A/O, A/A), regulatory MBL2 promoter polymorphism in position –221 (XX, XY, YY), anti-cyclic citrullinated peptide 2 antibodies (anti-CCP2), disease activity by Disease Activity Score-28 (DAS28 score), physical disability by Health Assessment Questionnaire (HAQ) score, and erosive changes in hands and feet (Sharp-van der Heijde score). RESULTS: Eight patients were homozygous MBL2 defective (O/O), 101 belonged to an intermediate group, and 49 were MBL2 high producers (YA/YA). Anti-CCP was present in 93 patients (59%). High scores of disease activity, C-reactive protein-based DAS28 (p = 0.02), and physical disability by HAQ (p = 0.01) were associated with high MBL2 expression genotypes in a gene-dose dependent way, but only in anti-CCP-positive patients. At this early stage of the disease there was no association with erosion score from radiographs. CONCLUSION: The results point to a synovitis-enhancing effect of MBL in anti-CCP-positive RA, whereas such an effect was not demonstrated for joint erosions.

Recently we found that the incidence of restenosis after carotid endarterectomy was significantly higher in patients homozygous for the normal genotype of mannose-binding lectin (MBL2) than in with patients with MBL2variant genotypes. Several growth factors are also known to contribute to restenosis. Therefore, we investigated whether early postoperative changes in serum vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and platelet-derived growth factor (PDGF) concentrations and MBL2genotypes interact in the development of restenosis.

Reduced levels of somatic hypermutation (SHM) have recently been described in IgG-switched immunoglobulin genes in a minority of patients with common variable immunodeficiency (CVID), demonstrating a disruption of the normal linkage between isotype switch and SHM. To see if, irrespective of isotype, there is a tendency to use unmutated immunoglobulin genes in CVID, we studied SHM in κ light-chain transcripts using a VκA27-specific restriction enzyme-based hot-spot mutation assay (IgκREHMA). Hot-spot mutations were found in 48% (median; reference interval, 28%-62%) of transcripts from 53 healthy controls. Values were significantly lower in 31 patients (median, 7.5%; range, 0%-73%; P < .0000001) of whom 24 (77%) had levels below the reference interval. Low levels of SHM correlated with increased frequency of severe respiratory tract infection (SRTI; P < .005), but not with diarrhea (P = .8). Mannose-binding lectin (MBL) deficiency also correlated with SRTI score (P = .009). However, the correlation of SHM and SRTI was also seen when only patients with normal MBL genotypes were analyzed (n = 18, P = .006). A slight decline of mutated fractions over years was noted (P = .01). This suggests that most patients with CVID fail to recruit affinity-maturated B cells, adding a qualitative deficiency to the quantitative deficiency characterizing these patients.

Reduced levels of somatic hypermutation (SHM) have recently been described in IgG-switched immunoglobulin genes in a minority of patients with common variable immunodeficiency (CVID), demonstrating a disruption of the normal linkage between isotype switch and SHM. To see if, irrespective of isotype, there is a tendency to use unmutated immunoglobulin genes in CVID, we studied SHM in κ light-chain transcripts using a VκA27-specific restriction enzyme-based hot-spot mutation assay (IgκREHMA). Hot-spot mutations were found in 48% (median; reference interval, 28%-62%) of transcripts from 53 healthy controls. Values were significantly lower in 31 patients (median, 7.5%; range, 0%-73%; P< .0000001) of whom 24 (77%) had levels below the reference interval. Low levels of SHM correlated with increased frequency of severe respiratory tract infection (SRTI; P< .005), but not with diarrhea (P= .8). Mannose-binding lectin (MBL) deficiency also correlated with SRTI score (P= .009). However, the correlation of SHM and SRTI was also seen when only patients with normal MBL genotypes were analyzed (n = 18, P= .006). A slight decline of mutated fractions over years was noted (P= .01). This suggests that most patients with CVID fail to recruit affinity-maturated B cells, adding a qualitative deficiency to the quantitative deficiency characterizing these patients.

Variant alleles in the mannose-binding lectin (MBL) gene (mbl2) causing low levels of functional MBL are associated with susceptibility to different infections and are common in areas where malaria is endemic. Therefore, we investigated whether MBL variant alleles in 551 children from Ghana were associated with the occurrence and outcome parameters of Plasmodium falciparum malaria and asked whether MBL may function as an opsonin for P. falciparum. No difference in MBL genotype frequency was observed between infected and noninfected children or between children with cerebral malaria and/or severe malarial anemia and children with uncomplicated malaria. However, patients with complicated malaria who were homozygous for MBL variant alleles had significantly higher parasite counts and lower blood glucose levels than their MBL-competent counterparts. Distinct calcium-dependent binding of MBL to the membrane of P. falciparum-infected erythrocytes, which could be inhibited by mannose, was observed. Further characterization revealed that MBL reacted with a P. falciparum glycoprotein identical to the 78-kDa glucose-regulated stress protein of P. falciparum. MBL seems to be a disease modifier in clinical malaria and to function as an opsonin for erythrocytes invaded by P. falciparum and may thus be involved in sequestration of the parasite, which in turn may explain the association between homozygosity for MBL variant alleles and high parasite counts.

ABSTRACTVariant alleles in the mannose-binding lectin (MBL) gene (mbl2) causing low levels of functional MBL are associated with susceptibility to different infections and are common in areas where malaria is endemic. Therefore, we investigated whether MBL variant alleles in 551 children from Ghana were associated with the occurrence and outcome parameters of Plasmodium falciparummalaria and asked whether MBL may function as an opsonin for P. falciparum. No difference in MBL genotype frequency was observed between infected and noninfected children or between children with cerebral malaria and/or severe malarial anemia and children with uncomplicated malaria. However, patients with complicated malaria who were homozygous for MBL variant alleles had significantly higher parasite counts and lower blood glucose levels than their MBL-competent counterparts. Distinct calcium-dependent binding of MBL to the membrane of P. falciparum-infected erythrocytes, which could be inhibited by mannose, was observed. Further characterization revealed that MBL reacted with a P. falciparumglycoprotein identical to the 78-kDa glucose-regulated stress protein of P. falciparum. MBL seems to be a disease modifier in clinical malaria and to function as an opsonin for erythrocytes invaded by P. falciparumand may thus be involved in sequestration of the parasite, which in turn may explain the association between homozygosity for MBL variant alleles and high parasite counts.

Epidemiological data indicate that acute lymphoblastic leukemia (ALL) could be induced by interactions between the immune system and early childhood infections. Mannose-binding lectin (MBL) plays a critical role in the immune response in early childhood before specific immune protection develops. We investigated whether there may be an association between childhood ALL and low-producing MBLgenotypes. Serum MBL levels depend on normal (A)or defective (O) alleles, and on normal (Y) or reduced (X) promoter activities. For this study, 137 noninfants with ALL and 250 controls were classified into 3 MBL genotype groups according to their influence on the serum level of functional MBL: group I, YA/YA andYA/XA (higher levels); group II, XA/XA andYA/O (intermediate levels); and group III, MBLinsufficiency with XA/O or O/O(MBL-deficient) genotypes. Compared with controls, cases more often had low-level genotypes (I/II/III: 63 [46%]/44 [32%]/30 [22%] vs 145 [58%]/65 [26%]/40 [16%];P = .02) and MBL deficiency (8.8% vs 2.8%;P = .009). Thus, the ALL odds ratio forMBL-deficient versus nondeficient individuals was 3.3 (95% CI, 1.3-8.7), whereas the ALL odds ratio for group I versus group II/III genotypes was 0.62 (95% CI, 0.41-0.94). MBL group III patients were significantly younger at diagnosis than patients in group I/II (median, 3.9 vs 5.2 years; P = .04). The study shows that the presence of low-level MBL genotypes is associated with an increased risk of childhood ALL, particularly with early age at onset.

Epidemiological data indicate that acute lymphoblastic leukemia (ALL) could be induced by interactions between the immune system and early childhood infections. Mannose-binding lectin (MBL) plays a critical role in the immune response in early childhood before specific immune protection develops. We investigated whether there may be an association between childhood ALL and low-producing MBLgenotypes. Serum MBL levels depend on normal (A)or defective (O) alleles, and on normal (Y) or reduced (X) promoter activities. For this study, 137 noninfants with ALL and 250 controls were classified into 3 MBLgenotype groups according to their influence on the serum level of functional MBL: group I, YA/YAandYA/XA(higher levels); group II, XA/XAandYA/O(intermediate levels); and group III, MBLinsufficiency with XA/Oor O/O(MBL-deficient) genotypes. Compared with controls, cases more often had low-level genotypes (I/II/III: 63 [46%]/44 [32%]/30 [22%] vs 145 [58%]/65 [26%]/40 [16%];P= .02) and MBL deficiency (8.8% vs 2.8%;P= .009). Thus, the ALL odds ratio forMBL-deficient versus nondeficient individuals was 3.3 (95% CI, 1.3-8.7), whereas the ALL odds ratio for group I versus group II/III genotypes was 0.62 (95% CI, 0.41-0.94). MBL group III patients were significantly younger at diagnosis than patients in group I/II (median, 3.9 vs 5.2 years; P= .04). The study shows that the presence of low-level MBLgenotypes is associated with an increased risk of childhood ALL, particularly with early age at onset.

OBJECTIVE: To determine whether variant alleles of the mannose-binding lectin (MBL) gene causing low serum concentrations of MBL and/or polymorphisms of HLA-DRB1 are associated with increased susceptibility to polymyalgia rheumatica (PMR) and giant cell arteritis (GCA) or particular clinical phenotypes of PMR/GCA. METHODS: MBL and HLA-DRB1 alleles were determined by polymerase chain reaction in 102 Danish patients with PMR (n = 37) or GCA (n = 65). Two hundred fifty and 193 healthy individuals served as controls for MBL and HLA genotyping, respectively. RESULTS: The prevalence of MBL variant alleles in controls, patients with PMR only, and patients with GCA was 37, 32, and 53% (p = 0.01), respectively. HLA-DRB1*04 was found in 47% of patients with PMR only and in 54% of patients with GCA, which differed significantly from the 35% found in controls (p = 0.01). HLA-DR4 alleles were not associated with any clinical phenotypes of PMR/GCA, whereas MBL variant alleles were associated with cranial arteritis, high erythrocyte sedimentation rate, and low B-hemoglobin. CONCLUSION: We found MBL variant alleles and HLA-DR4 alleles to be weak susceptibility markers for GCA. In patients with PMR/GCA, MBL variant alleles were associated with signs of increased inflammatory activity and clinical signs of arteritic manifestations. This was not found for HLA-DR4 alleles. These findings indicate that HLA-DR4 and MBL are contributing to the pathophysiology of GCA at different levels in the disease process.

To investigate the possible association of mannose-binding lectin (MBL) genotypes with the outcome of rheumatoid arthritis (RA). MBL genotypes and plasma concentrations were retrospectively determined in 140 RA patients who were selected from a major cohort followed up prospectively for up to 32 years. MBL-insufficient patients (those with 2 defective structural MBL alleles or with 1 defective allele combined with a low-expression variant of the normal allele) had unfavorable outcomes. The relative risk of a severe radiographic outcome event (30% of maximum radiographic destruction, or an RE30) was 3.1 (95% confidence interval 1.8–5.1) in the MBL-insufficient group versus the MBL-competent group (P &lt; 0.0001). An RE30 occurred in 50% of MBL-competent patients within 17 years, while such an event occurred 9 years earlier in MBL-insufficient patients (i.e., within 8 years) (P &lt; 0.0001). During the first 15 years, there was a significant trend toward lower hemoglobin levels (P &lt; 0.04), higher erythrocyte sedimentation rates (P &lt; 0.02), and a higher number of swollen joints (P &lt; 0.05) in the MBL-insufficient group. MBL genotypes giving rise to MBL insufficiency are highly significant risk factors for fast progression of radiographic joint destruction.