l-ficolin-MASP arm of the complement system in schizophrenia.

The abnormal neurodevelopment secondary to in utero adversities, such as hypoxia, malnutrition and maternal infections, underlies schizophrenia (SZ) etiology. As the genes of MBL-associated serine proteases (MASP) of the complement lectin pathway, MASP1 and MASP2, are expressed in the developing cortex and are functionally important for neuronal migration, we hypothesize that the malfunction ofl-ficolin-MASP arm may also be involved in schizophrenia pathophysiology as it was shown for MBL-MASP complexes. We investigated serum l-ficolin and plasma MASP-2 levels, the activity of l-ficolin-bound MASP-2, as well as an array of the complement-related variables in chronic schizophrenic patients in the acute phase of the disease and controls without physical or mental diagnoses. The median concentration of l-ficolin in Armenian controls was 3.66 µg/ml and similar to those reported for other Caucasian populations. SZ-cases had âˆ¼40 % increase in serum l-ficolin (median 5.08 µg/ml; P < 0.0024). In the pooled sample, l-ficolin level was higher in males than in females (P < 0.0031), but this gender dichotomy was not affecting the variable association with schizophrenia (P < 0.016). Remarkably, MASP-2 plasma concentration showed gender-dependent significant variability in the group of patients but not in controls. When adjusted for gender and gender*diagnosis interaction, a significantly high MASP-2 level in female patients versus female controls was observed (median: 362 ng/ml versus 260 ng/ml, respectively; P < 0.0020). A significant increase in l-ficolin-bound MASP-2 activity was also observed in schizophrenia (on the median, cases vs controls: 7.60 vs 6.50 RU; P < 0.021). Correlation analyses of the levels of l-ficolin and MASP-2, l-ficolin-(MASP-2) activity and the demographic data did not show any significant association with the age of individuals, family history, age at onset and duration of the illness, and smoking. Noteworthy, the levels of l-ficolin and MASP-2 in circulation were significantly associated with the type of schizophrenia (paranoid SZ-cases had much higher l-ficolin (P < 0.0035) and lower MASP-2 levels than the other types combined (P < 0.049)). Correlations were also found between: (i) the classical pathway functional activity and l-ficolin level (rs = 0.19, P < 0.010); (ii) the alternative pathway functional activity and MASP-2 level (rs = 0.26, P < 0.00035); (iii) the activity of l-ficolin-bound MASP2 and the downstream C2 component haemolytic activity (rs = -0.19, P < 0.017); and (iv) l-ficolin and the upstream C-reactive protein (CRP) serum concentrations (r = 0.28, P < 0.018). Overall, the results showed l-ficolin-related lectin pathway alterations in schizophrenia pathophysiology. It is likely that in addition to the MBL-MASP component over-activity reported previously, the alterations of the lectin pathway in schizophrenia also involve variations of l-ficolin-(MASP-2) on protein concentration and activity levels.

Pattern Recognition Molecules of Lectin Complement Pathway in Ischemic Stroke.

PURPOSE: The current study aimed to investigate in an Armenian population the levels of pattern recognition molecules (PRMs) of lectin complement pathway (LCP), MBL (mannan-binding lectin) and M-ficolin in plasma in ischemic stroke (IS), and the possible association of 11 single nucleotide polymorphisms (SNPs) in MBL2, FCN1 and FCN2 genes. PATIENTS AND METHODS: A total of 122 patients with IS and 150 control subjects were included in this study. Immunofluorometric assays (TRIFMAs) and real-time polymerase chain reactions with TaqMan probes were conducted. RESULTS: According to the results, the levels of M-ficolin in IS patients are significantly higher than in control subjects, and the MBL2 rs11003125 and rs12780112 SNPs, as well as MBL2 rs12780112*T and FCN1 rs10120023*T minor alleles (MAs) are negatively associated with the risk of IS. Further, MBL2 rs11003125 and rs1800450 SNPs and the carriage of their MAs, as well as FCN1 rs2989727 SNP and the carriage of FCN1 rs10120023*T MA significantly alter plasma MBL and M-ficolin levels in IS patients, respectively. Five common haplotypes in MBL2 gene and three common haplotypes in FCN1 and FCN2 genes were revealed, among which CGTC was negatively associated with IS and decreasing MBL plasma levels in IS. CONCLUSION: In conclusion, we suggest that LCP PRMs are associated with the risk of developing IS, and may also participate in pathological events leading to post-ischemic brain damage. This study emphasizes the important contribution of alterations of LCP PRMs on genomic and proteomic levels to the pathomechanisms of ischemic stroke, at least in an Armenian population.

C3dg Quantification by PEG Precipitation and or TRIFMA.

Detection of complement activation products can be carried out in a number of ways, and different methods are used in different laboratories. No international standard for measuring complement activation in the clinical setting has been agreed upon.Here we describe a modified assay for measuring C3dg. The assay is simple, inexpensive and stable. The estimation of C3dg directly reflects complement turnover independently of activation pathway.

The human natural anti-αGal antibody targets common pathogens by broad-spectrum polyreactivity.

Naturally occurring antibodies are abundant in human plasma, but their importance in the defence against bacterial pathogens is unclear. We studied the role of the most abundant of such antibodies, the antibody against terminal galactose-α-1,3-galactose (anti-αGal), in the protection against pneumococcal infections (Streptococcus pneumonia). All known pneumococcal capsular polysaccharides lack terminal galactose-α-1,3-galactose, yet highly purified human anti-αGal antibody of the IgG class reacted with 48 of 91 pneumococcal serotypes. Anti-αGal was found to contain multiple antibody subsets that possess distinct specificities beyond their general reactivity with terminal galactose-α-1,3-galactose. These subsets in concert targeted a wide range of microbial polysaccharides. We found that anti-αGal constituted up to 40% of the total antibody reactivity to pneumococci in normal human plasma, that anti-αGal drives phagocytosis of pneumococci by human neutrophils and that the anti-αGal level was twofold lower in patients prone to pneumococcal infections compared with controls. Moreover, during a 48-year period in Denmark, the 48 anti-αGal-reactive serotypes caused fewer invasive pneumococcal infections (n = 10 927) than the 43 non-reactive serotypes (n = 18 107), supporting protection on the population level. Our findings explain the broad-spectrum pathogen reactivity of anti-αGal and support that these naturally occurring polyreactive antibodies contribute significantly to human protective immunity.

Associations of ficolins and mannose-binding lectin with acute myeloid leukaemia in adults.

We investigated clinical associations of ficolins and mannose-binding lectin (MBL) in 157 patients suffering from acute myeloid leukaemia (AML). Concentrations of ficolin-1, ficolin-2, ficolin-3 and MBL (before chemotherapy) in serum were determined as were selected polymorphisms of the corresponding genes (FCN1, FCN2, FCN3 and MBL2). The control group (C) consisted of 267 healthy unrelated individuals. Median level of ficolin-1 in patients was lower (p < 0.000001) while median levels of ficolin-2, ficolin-3 and MBL were higher (p < 0.000001, p < 0.000001 and p = 0.0016, respectively) compared with controls. These findings were generally associated with AML itself, however the highest MBL levels predicted higher risk of severe hospital infections (accompanied with bacteremia and/or fungaemia) (p = 0.012) while the lowest ficolin-1 concentrations tended to be associated with prolonged (> 7 days) fever (p = 0.026). Genotyping indicated an association of G/G homozygosity (corresponding to FCN1 gene - 542 G > A polymorphism) with malignancy [p = 0.004, OR = 2.95, 95% CI (1.41-6.16)]. Based on ROC analysis, ficolin-1, -2 and -3 may be considered candidate supplementary biomarkers of AML. Their high potential to differentiate between patients from non-malignant controls but also from persons suffering from other haematological cancers (multiple myeloma and lymphoma) was demonstrated.

Complement activation by human IgG antibodies to galactose-α-1,3-galactose.

Some human antibodies may paradoxically inhibit complement activation on bacteria and enhance pathogen survival in humans. This property was also claimed for IgG antibodies reacting with terminal galactose-α-1,3-galactose (Galα3Gal; IgG anti-αGal), a naturally occurring and abundant antibody in human plasma that targets numerous different pathogens. To reinvestigate these effects, we used IgG anti-αGal affinity isolated from a pool of normal human IgG and human hypogammaglobulinaemia serum as a complement source. Flow cytometry was performed to examine antibody binding and complement deposition on pig erythrocytes, Escherichia coli O86 and Streptococcus pneumoniae serotype 9V. Specific nanobodies were used to block the effect of single complement factors and to delineate the complement pathways involved. IgG anti-αGal was capable of activating the classical complement pathway on all the tested target cells. The degree of activation was exponentially related to the density of bound antibody on E. coli O86 and pig erythrocytes, but more linearly on S. pneumoniae 9V. The alternative pathway of complement amplified complement deposition. Deposited C3 fragments covered the activating IgG anti-αGal, obstructing its detection and highlighting this as a likely general caveat in studies of antibody density and complement deposition. The inherent capacity for complement activation by the purified carbohydrate reactive IgG anti-αGal was similar to that of normal human IgG. We propose that the previously reported complement inhibition by IgG anti-αGal relates to suboptimal assay configurations, in contrast to the complement activating property of the antibodies demonstrated in this paper.

Adding MASP1 to the lectin pathway-Leprosy association puzzle: Hints from gene polymorphisms and protein levels.

BACKGROUND: Deposition of complement factors on Mycobacterium leprae may enhance phagocytosis. Such deposition may occur through the lectin pathway of complement. Three proteins of the lectin pathway are produced from the gene MASP1: Mannan-binding lectin-associated serine protease 1 (MASP-1) and MASP-3 and mannan-binding lectin-associated protein of 44 kDa (MAp44). Despite their obvious importance, the roles played by these proteins have never been investigated in leprosy disease. METHODOLOGY: We haplotyped five MASP1 polymorphisms by multiplex sequence-specific PCR (intronic rs7609662*G>A and rs13064994*C>T, exon 12 3'-untranslated rs72549262*C>G, rs1109452*C>T and rs850314*G>A) and measured MASP-1, MASP-3 and MAp44 serum levels in 196 leprosy patients (60%, lepromatous) and 193 controls. PRINCIPAL FINDINGS: Lower MASP-3 and MAp44 levels were observed in patients, compared with controls (P = 0.0002 and P<0.0001, respectively) and in lepromatous, compared with non-lepromatous patients (P = 0.008 and P = 0.002, respectively). Higher MASP-3 levels were present in controls carrying variants/haplotypes associated with leprosy resistance (rs13064994*T, rs1109452_rs850314*CG within GT_CCG and rs850314*A: OR = 0.5-0.6, Pcorr = 0.01-0.04). Controls with rs1109452*T, included in susceptibility haplotypes (GT_GTG/GT_CTG: OR = 2.0, Pcorr = 0.03), had higher MASP-1 and lower MASP-3 levels (P≤0.009). Those with GC_CCG, presented increasing susceptibility (OR = 1.7, Pcorr = 0.006) and higher MAp44 levels (P = 0.015). MASP-3 expression decreased in patients, compared with controls carrying rs1109452_rs850314*CA or CG (P≤0.02), which may rely on exon 12 CpG methylation and/or miR-2861/miR-3181 mRNA binding. CONCLUSION: Polymorphisms regulating MASP-3/MAp44 availability in serum modulate leprosy susceptibility, underlining the importance of lectin pathway regulation against pathogens that exploit phagocytosis to parasitize host macrophages.

Abundant human anti-Galα3Gal antibodies display broad pathogen reactivity.

Antibodies of the IgG class to terminal Galα3Gal (IgG anti-αGal) is abundant in human plasma and are reported to bind most sepsis-causing Gram-negative bacteria. However, these seminal findings, made more than two decades ago, have not been reexamined. Our aim was to assess IgG anti-αGal´s pathogen reactivity. We affinity purified IgG anti-αGal from a therapeutic grade normal human IgG pool applying two rounds of positive selection with Galα3Gal-coupled beads and included removal of column matrix reactive antibodies. The purified antibodies were rigorously characterized in terms of specificity and purity in various solid-phase immunoassays. We used flow cytometry to study reactivity against 100 consecutive clinical isolates diagnosed as cause of sepsis in humans. We found that the purified IgG anti-αGal displays high specificity for Galα3Gal. Also, IgG anti-αGal at 5 mg/L bound 56 out of 100 pathogens with predilection for Gram-positive bacteria binding 39 out of 52 strains. We confirm that although IgG anti-αGal comprise a small fraction of the human antibody pool (~0.1%), these antibodies targets an impressively large part of pathogens causing invasive disease.

Selected factors of the innate immunity in Polish patients suffering from pulmonary tuberculosis.

We conducted a prospective study of 453 Polish patients suffering from pulmonary tuberculosis and 267 healthy controls. Selected polymorphisms of the genes encoding for collectins, ficolins and MBL-associated serine protease 2 were investigated as were serum concentrations of mannose-binding lectin, surfactant protein D, ficolin-1 and ficolin-3. The number of MBL2 gene exon 1 variant allele carriers was significantly higher in patients, compared with controls. The homozygosity for SFTPA2 +26 C > A SNP variant allele occurred less commonly within TB, while homozygosity for the FCN1 -542 G > A major allele was less frequent within the control group. Two patients were found MASP-2-deficient. Serum concentrations of MBL, SP-D and ficolin-1 were higher amongst patients while the converse was found for ficolin-3. Ficolin-1 had high specificity to differentiate between individuals with tuberculosis and healthy persons and therefore may be considered potential disease marker.

Factors involved in initiation and regulation of complement lectin pathway influence postoperative outcome after pediatric cardiac surgery involving cardiopulmonary bypass.

Congenital heart disease (CHD) often requires surgical intervention, and is sometimes associated with life-threatening post-operative complications. We have investigated some factors of the innate immune system involved in the initiation or regulation of complement lectin pathway activation (MASP-1, MASP-2 MASP-3, MAp19, MAp44, ficolin-3) and related them to complications and prognosis in 190 pediatric patients undergoing CHD repair with the use of cardiopulmonary bypass (CPB). Patients with MAp44 levels ≤1.81 µg/ml more frequently experienced low cardiac output syndrome (LCOS), renal insufficiency, systemic inflammatory response syndrome (SIRS) and multiorgan dysfunction (MODS). Low MASP-3 (≤5.18 µg/ml) and high MASP-1 (≥11.7 µg/ml) levels were often associated with fatal outcome. Low ficolin-3 concentrations (≤10.1 µg/ml) were more common among patients experiencing SIRS and MODS than in those without complications. However, patients suffering from SIRS and MODS with low ficolin-3 had a much better prognosis (91% survival vs. 37% among other patients; p = 0.007). A discriminating value of 12.7 µg/ml ficolin-3 yielded 8% vs. 60% mortality (p = 0.001). Our data extend the knowledge concerning involvement of proteins of the lectin pathway in development of post-CPB complications. The potential prognostic value of low preoperative MAp44 and high preoperative ficolin-3 seems promising and warrants independent confirmation.

Associations of Ficolins With Hematological Malignancies in Patients Receiving High-Dose Chemotherapy and Autologous Hematopoietic Stem Cell Transplantations.

A prospective study of 312 patients [194 with multiple myeloma (MM) and 118 with lymphomas (LYMPH)] receiving high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto-HSCT) was conducted. Ficolins are innate immune defense factors, able to distinguish between "self" "abnormal self," and "non-self" and contribute to the elimination of the last two by direct opsonization and/or initiation of complement activation via the lectin pathway. Concentrations of ficolin-1, ficolin-2, and ficolin-3 in serially taken serum samples were determined as were the polymorphisms of the corresponding (FCN1, FCN2, and FCN3) genes. Serum samples were collected before conditioning chemotherapy, before HSCT, and once weekly post-HSCT (four to five samples in total); some patients were also sampled at 1 and/or 3 months post-transplantation. The control group (C) consisted of 267 healthy unrelated individuals. Median ficolin-1 and ficolin-2 (but not ficolin-3) levels in MM patients' sera taken before chemotherapy were lower (and correspondingly frequencies of the lowest concentrations were higher) compared with controls. That appeared to be associated with the malignant disease itself rather than with post-HSCT complications (febrile neutropenia, infections accompanied, or not with bacteremia). Higher frequencies of the FCN1 genotype G/A-C/C-G/G (corresponding to polymorphisms at positions -542, -144, and +6658, respectively) and FCN2 gene heterozygosity for the -857 C>A polymorphism were found among patients diagnosed with MM compared with the C group. Furthermore, FCN2 G/G homozygosity (-557 A>G) was found more frequently and heterozygosity G/T at +6424 less frequently among LYMPH patients than among the healthy subjects. Heterozygosity for +1637delC mutation of the FCN3 gene was more common among patients diagnosed with lymphomas who experienced hospital infections. Although no evidence for an association of low ficolin-1 or ficolin-2 with infections during neutropenia following chemotherapy before HSCT was found, we observed a possible protective effect of ficolins during follow-up.

MASP-1 and MASP-2 Serum Levels Are Associated With Worse Prognostic in Cervical Cancer Progression.

Background: MBL-associated serine proteases (MASP-1, MASP-2, MASP-3, MAp-44, and MAp-19) are key factors in the activation of the lectin pathway of complement. Serum levels of these components have been associated with recurrence and poor survival of some types of cancer, such as colorectal and ovarian cancer. In this investigation, we determined the serum levels of MASP-1, MASP-2, MASP-3, MAp-44, and MAp-19 in patients with cervical cancer and cervical intraepithelial neoplasia (CIN). Methods:A total of 351 women who underwent screening for cervical cancer or treatment at the Erasto Gaertner Cancer Hospital in Curitiba-Brazil, were enrolled in the study. Based on their latest cervical colposcopy-guided biopsy results, they were divided into four groups: CIN-I: n = 52; CIN-II: n = 73; CIN-III: n = 141; and invasive cancer: n = 78. All the serum protein levels were determined by time-resolved immunofluorometric assay (TRIFMA). Results:Patients with invasive cancer presented significantly higher MASP-2, MASP-1, and MAp-19 serum levels than other groups (p < 0.0001; p = 0.012; p = 0.025 respectively). No statistically significant differences in MASP-3 and MAp-44 serum levels were found between the four studied groups. In addition, high MASP-2, MASP-1, and MAp-19 serum levels were significantly associated with poor survival in patients with invasive cancer and relapse (p = 0.002, p = 0.0035 and p = 0.025, respectively). Conclusion:High MASP-2, MASP-1, and MAp-19 serum levels were associated with cervical cancer progression and worse disease prognosis. These novel findings demonstrate the involvement of the serine proteases of the lectin pathway in the pathogenesis of cervical cancer and future investigations should clarify their role in the disease process.

Ficolin-1 and Ficolin-3 Plasma Levels Are Altered in HIV and HIV/HCV Coinfected Patients From Southern Brazil.

The complement system is a key component of the innate immune system, participating in the surveillance against infectious agents. Once activated by one of the three different pathways, complement mediates cell lysis, opsonization, signalizes pathogens for phagocytosis and induces the adaptive immune response. The lectin pathway is constituted by several soluble and membrane bound proteins, called pattern recognition molecules (PRM), including mannose binding lectin (MBL), Ficolins-1, -2, and -3, and Collectin 11. These PRMs act on complement activation as recognition molecules of pathogen-associated molecular patterns (PAMPs) such as N-acetylated, found in glycoproteins of viral envelopes. In this study, Ficolin-1 and Ficolin-3 plasma levels were evaluated in 178 HIV patients (93 HIV; 85 HIV/HCV) and 85 controls from southern Brazil. Demographic and clinical-laboratory findings were obtained during medical interview and from medical records. All parameters were assessed by logistic regression, adjusted for age, ancestry, and sex. Significantly lower levels of Ficolin-1 were observed in HIV/HCV coinfected when compared to HIV patients (p = 0.005, median = 516 vs. 667 ng/ul, respectively) and to controls (p < 0.0001, 1186 ng/ul). Ficolin-1 levels were lower in males than in females among HIV patients (p = 0.03) and controls (p = 0.0003), but no association of Ficolin-1 levels with AIDS was observed. On the other hand, Ficolin-3 levels were significantly lower in controls when compared to HIV (p < 0.0001, medians 18,240 vs. 44,030 ng/ml, respectively) and HIV/HCV coinfected (p < 0.0001, 40,351 ng/ml) patients. There was no correlation between Ficolin-1 and Ficolin-3 levels and age, HIV viral load or opportunistic infections. However, Ficolin-3 showed a positive correlation with T CD4 cell counts in HIV monoinfected patients (p = 0.007). We provide here the first assessment of Ficolin-1 and-3 levels in HIV and HIV/HCV coinfected patients, which indicates a distinct role for these pattern recognition molecules in both viral infections.

The Role of Complement Activating Collectins and Associated Serine Proteases in Patients With Hematological Malignancies, Receiving High-Dose Chemotherapy, and Autologous Hematopoietic Stem Cell Transplantations (Auto-HSCT).

We conducted a prospective study of 312 patients (194 with multiple myeloma, 118 with lymphomas) receiving high-dose conditioning chemotherapy and autologous hematopoietic stem cell transplantation (auto-HSCT). Polymorphisms of MBL2 and MASP2 genes were investigated and serial measurements of serum concentrations of mannose-binding lectin (MBL), CL-LK collectin and MASP-2 as well as activities of MBL-MASP-1 and MBL-MASP-2 complex were made. Serum samples were taken before conditioning chemotherapy, before HSCT and once weekly after (totally 4-5 samples); in minority of subjects also 1 and/or 3 months post transplantation. The results were compared with data from 267 healthy controls and analyzed in relation to clinical data to explore possible associations with cancer and with chemotherapy-induced medical complications. We found a higher frequency of MBL deficiency-associated genotypes (LXA/O or O/O) among multiple myeloma patients compared with controls. It was however not associated with hospital infections or post-HSCT recovery of leukocytes, but seemed to be associated with the most severe infections during follow-up. Paradoxically, high MBL serum levels were a risk factor for prolonged fever and some infections. The first possible association of MBL2 gene 3'-untranslated region polymorphism with cancer (lymphoma) in Caucasians was noted. Heterozygosity for MASP2 gene +359 A>G mutation was relatively frequent in lymphoma patients who experienced bacteremia during hospital stay. The median concentration of CL-LK was higher in myeloma patients compared with healthy subjects. Chemotherapy induced marked increases in serum MBL and MASP-2 concentrations, prolonged for several weeks and relatively slighter decline in CL-LK level within 1 week. Conflicting findings on the influence of MBL on infections following chemotherapy of myeloma and lymphoma have been reported. Here we found no evidence for an association between MBL deficiency and infection during the short period of neutropenia following conditioning treatment before HSCT. However, we noted a possible protective effect of MBL during follow-up, and suspected that to be fully effective when able to act in combination with phagocytic cells after their recovery.

The Lectin Pathway of Complement Activation in Patients with Systemic Lupus Erythematosus.

OBJECTIVE: The pathogenesis of systemic lupus erythematosus (SLE) involves complement activation. Activation of complement through the classical pathway (CP) is well established. However, complement activation through pattern recognition not only happens through the CP, but also through the lectin pathway (LP). We investigated the hypothesis that the LP is activated in SLE and involved in the pathogenesis of the disease. METHODS: Using immunoassays developed in-house, we measured concentrations of LP proteins in a cohort of 372 patients with SLE and 170 controls. We estimated complement activation measuring total C3, and investigated whether LP protein concentrations were associated with complement activation and disease activity. Protein changes and disease activity over time were assessed in a cohort of 52 patients with SLE followed with repeated samples over a 5-year period. RESULTS: Concentrations of LP proteins in SLE were altered compared with controls. The differences observed in LP proteins associated with complement activation were reflected by a decrease in total C3. The pattern recognition molecules (M-ficolin, CL-L1, and CL-K1), the serine protease (MASP-3), and the associated protein (MAp19) displayed a negative correlation with disease activity. Changes in MASP-2 concentrations over time correlated significantly with increased disease activity. Association between active proteinuria and serum concentration was observed for MASP-3 and MAp19. CONCLUSION: In patients with SLE, we measured specific changes in LP proteins that are associated with complement activation and disease activity, indicating that the LP is activated in patients with SLE. These novel findings substantiate the involvement of the LP in SLE.

The C3dg Fragment of Complement Is Superior to Conventional C3 as a Diagnostic Biomarker in Systemic Lupus Erythematosus.

Introduction/objectives: In 2012, hypocomplementemia was included in the classification criteria of systemic lupus erythematosus (SLE). The suggested measurement of C3 or C4 often reflect disease activity poorly. Our objective was to establish an assay measuring C3dg, which is generated following complement activation, and to evaluate the assay in a cross-sectional SLE cohort. Method: We included SLE patients (n = 169) and controls (n = 170) and developed a modified C3dg assay where C3dg fragments were separated from the large plasma proteins by polyethylene glycol (PEG), and the supernatant containing the C3dg fragment was used for analysis in an antibody-based sandwich-type assay. Gel permeation chromatography and western blotting were used to establish the optimal conditions for PEG precipitation. Results: 16% PEG was optimal for separating C3dg from C3 and the larger protein fragments. The assay showed a high degree of stability when using EDTA plasma, and measurements correlated well with commercially available complement activation assays. SLE patients had higher concentrations in plasma of C3dg than controls (p < 0.05). ROC analysis showed that the C3dg activation fragment of C3 with an AUC of 0.96 (CI 0.94-0.98) was superior to C3 (AUC 0.52) in differentiating between patients and controls. Conclusion: Our results present a modified assay for the measurement of C3dg. We demonstrate that C3dg was superior to conventional C3 measurements in discriminating SLE patients from controls. We suggest that C3dg should be considered as a complement activation measurement in the SLE classification criteria.

Endogenous Natural Complement Inhibitor Regulates Cardiac Development.

Congenital heart defects are a major cause of perinatal mortality and morbidity, affecting >1% of all live births in the Western world, yet a large fraction of such defects have an unknown etiology. Recent studies demonstrated surprising dual roles for immune-related molecules and their effector mechanisms during fetal development and adult homeostasis. In this article, we describe the function of an endogenous complement inhibitor, mannan-binding lectin (MBL)-associated protein (MAp)44, in regulating the composition of a serine protease-pattern recognition receptor complex, MBL-associated serine protease (MASP)-3/collectin-L1/K1 hetero-oligomer, which impacts cardiac neural crest cell migration. We used knockdown and rescue strategies in zebrafish, a model allowing visualization and assessment of heart function, even in the presence of severe functional defects. Knockdown of embryonic expression of MAp44 caused impaired cardiogenesis, lowered heart rate, and decreased cardiac output. These defects were associated with aberrant neural crest cell behavior. We found that MAp44 competed with MASP-3 for pattern recognition molecule interaction, and knockdown of endogenous MAp44 expression could be rescued by overexpression of wild-type MAp44. Our observations provide evidence that immune molecules are centrally involved in the orchestration of cardiac tissue development.

Structure and activation of C1, the complex initiating the classical pathway of the complement cascade.

The complement system is an important antimicrobial and inflammation-generating component of the innate immune system. The classical pathway of complement is activated upon binding of the 774-kDa C1 complex, consisting of the recognition molecule C1q and the tetrameric protease complex C1r2s2, to a variety of activators presenting specific molecular patterns such as IgG- and IgM-containing immune complexes. A canonical model entails a C1r2s2 with its serine protease domains tightly packed together in the center of C1 and an intricate intramolecular reaction mechanism for activation of C1r and C1s, induced upon C1 binding to the activator. Here, we show that the serine protease domains of C1r and C1s are located at the periphery of the C1r2s2 tetramer both when alone or within the nonactivated C1 complex. Our structural studies indicate that the C1 complex adopts a conformation incompatible with intramolecular activation of C1, suggesting instead that intermolecular proteolytic activation between neighboring C1 complexes bound to a complement activating surface occurs. Our results rationalize how a multitude of structurally unrelated molecular patterns can activate C1 and suggests a conserved mechanism for complement activation through the classical and the related lectin pathway.

Collectins, H-ficolin and LL-37 reduce influence viral replication in human monocytes and modulate virus-induced cytokine production.

Infiltrating activated monocytes are important mediators of damaging inflammation during influenza A virus (IAV) infection. We show that soluble respiratory proteins [collectins, surfactant proteins D (SP-D) and mannose binding lectin (MBL), H-ficolin and LL-37] inhibit replication of seasonal IAV in human monocytes. The collectins and H-ficolin also increased viral uptake by the cells, while LL-37 did not. H-ficolin was able to inhibit replication of the 2009 pandemic H1N1 strain (Cal09) in monocytes, but SP-D and LL-37 had significantly fewer inhibitory effects on this strain than on seasonal IAV. All of these proteins reduced IAV-induced TNF-α production, even in instances when viral replication was not reduced. We used modified recombinant versions of SP-D, MBL and ficolin to elucidate mechanisms through which these proteins alter monocyte interactions with IAV. We demonstrate the importance of the multimeric structure, and of binding properties of the lectin domain, in mediating antiviral and opsonic activity of the proteins. Hence, soluble inhibitors present in airway lining fluid may aid clearance of IAV by promoting monocyte uptake of the virus, while reducing viral replication and virus-induced TNF-α responses in these cells. However, SP-D and LL-37 have reduced ability to inhibit replication of pandemic IAV in monocytes.