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.

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.

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.

Influence of mannan-binding lectin and MAp44 on outcome in comatose survivors of out-of-hospital cardiac arrest.

AIM: The lectin complement pathway, initiated by mannan-binding-lectin (MBL) plays a role in tissue destruction following ischemia/reperfusion, and MBL deficiency has been associated with favorable outcome in stroke patients. MAp44 is produced in the heart and may theoretically function as an endogenous inhibitor of MBL-mediated activities. The aim of this study was to investigate the possible association between MBL deficiency, MAp44 levels and outcome in comatose survivors of out-of-hospital cardiac arrest (OHCA). METHODS: In a single center post hoc analysis of the prospective multicenter randomized Target Temperature Management (TTM) trial, we measured MBL and MAp44 levels at baseline, 24, 48 and 72 h after OHCA in 169 consecutive patients randomly assigned to TTM at 33 °C or 36 °C for 24h. Primary outcome was 30 days mortality and secondary outcome was favorable neurological outcome assessed by Cerebral Performance Category (CPC1-2) and modified Rankin Scale (mRS0-3) 180 days after OHCA. RESULTS: Patients with MBL deficiency (defined as plasma levels ≤100 ngml(-1) at baseline) (n=22) carried a 30-day mortality of 41% compared to 32% in MBL sufficient patient (n=147), p=0.55. Baseline MAp44 levels were not associated with mortality, p=0.25. There was no significant difference in neurological outcome between the two MBL groups assessed by CPC (p=0.69) and mRS (p=0.91). In multivariable models, baseline MBL (OR=1.0, p=0.70), (OR=1.5, p=0.30) and MAp44 levels (OR=1.0, p=0.99), (OR=1.6, p=0.21) were not associated with favorable neurological outcome assessed by CPC and mRS, respectively. CONCLUSIONS: In comatose survivors after cardiac arrest, neither MBL deficiency nor levels of MBL and MAp44 were associated with mortality or neurological outcome.

Investigation of Complement-activating Pattern Recognition Molecules and Associated Enzymes as Possible Inflammatory Markers in Oligoarticular and Systemic Juvenile Idiopathic Arthritis.

OBJECTIVE: The complement system plays a crucial role in the pathogenesis of inflammatory processes. The lectin pathway of the complement system is activated through the recognition of pathogens by soluble pattern recognition molecules (PRM), i.e., mannan-binding lectin (MBL), collectin-LK, and the ficolins. PRM are reportedly correlated to disease activity in rheumatoid arthritis (RA). The aim was to evaluate the pathogenic role of PRM in juvenile idiopathic arthritis (JIA). METHODS: We measured MBL, M-ficolin, H-ficolin, MBL-associated serine proteases (MASP) 1, MASP-2, MASP-3, and 2 alternative splice products, MBL-associated protein (MAp) 44 and MAp19, in plasma and synovial fluid (SF) of children with persistent oligoarticular (n = 109 in plasma, n = 38 in SF) and systemic JIA (n = 19 in plasma, n = 11 in SF). The concentrations of the proteins were measured by in-house time-resolved immunofluorometric assays. RESULTS: We observed significantly higher levels of M-ficolin, MASP-1, MASP-2, and MASP-3 in plasma and SF from patients with systemic JIA compared with persistent oligoarticular JIA (p < 0.001). In paired samples of plasma and SF from 47 patients with oligoarticular and systemic JIA, we observed higher concentrations in plasma for both subtypes for 7 of the measured proteins while the reverse relationship was observed for MASP-3. M-ficolin and MASP-2 correlated to erythrocyte sedimentation rate, C-reactive protein, white blood cell count, and platelet count (p < 0.001). M-ficolin was in addition related to the number of active joints and inversely related to hemoglobin levels. CONCLUSION: Our results point to plasma M-ficolin and MASP-2 as inflammatory markers in JIA. The levels of all proteins are higher in plasma than in SF, except for MASP-3, indicating that MASP-3 may be produced locally in joints.

Levels in plasma of the serine proteases and associated proteins of the lectin pathway are altered in patients with systemic lupus erythematosus.

OBJECTIVE: To examine whether proteins of the lectin pathway of the complement system are involved in systemic lupus erythematosus (SLE) pathogenesis. METHODS: Using time-resolved immunofluorometric assays, plasma levels of mannan-binding lectin (MBL)-associated serine proteases 1 (MASP-1), MASP-2, MASP-3, MBL-associated protein of 19 kDa (MAp19), and MAp44 were determined in 58 patients with SLE and 65 healthy controls (HC). RESULTS: Plasma concentrations in patients with SLE were higher than HC regarding MASP-1, MASP-3, and MAp44 (p < 0.0001, 0.0003, and 0.0013). Complement factor 3 correlated negatively and anti-dsDNA positively with levels of MAp19 (p = 0.0035, p = 0.0133). CONCLUSION: In SLE, plasma levels of MASP and MAp are altered and associated with SLE characteristics, supporting a role in SLE pathogenesis.

Structural insights into the initiating complex of the lectin pathway of complement activation.

The proteolytic cascade of the complement system is initiated when pattern-recognition molecules (PRMs) bind to ligands, resulting in the activation of associated proteases. In the lectin pathway of complement, the complex of mannan-binding lectin (MBL) and MBL-associated serine protease-1 (MASP-1) initiates the pathway by activating a second protease, MASP-2. Here we present a structural study of a PRM/MASP complex and derive the overall architecture of the 450 kDa MBL/MASP-1 complex using small-angle X-ray scattering and electron microscopy. The serine protease (SP) domains from the zymogen MASP-1 dimer protrude from the cone-like MBL tetramer and are separated by at least 20 nm. This suggests that intracomplex activation within a single MASP-1 dimer is unlikely and instead supports intercomplex activation, whereby the MASP SP domains are accessible to nearby PRM-bound MASPs. This activation mechanism differs fundamentally from the intracomplex initiation models previously proposed for both the lectin and the classical pathway.

Reibergrama para evaluar la síntesis intratecal de Lectina de unión a Manosa / Reibergram to evaluate the Mannan binding Lectin intrathecal synthesis

INTRODUCCIÓN: la dinámica particular de las proteínas derivadas del cerebro en el líquido cefalorraquídeo es diferente a la dinámica de las proteínas derivadas de la sangre. OBJETIVO: describir los datos empíricos de la lectina de unión a manosa y brindar una interpretación teórica de la dinámica de esta proteína a través de la confección un nuevo reibergrama. MÉTODOS: la lectina de unión a manosa en suero y líquido cfalorraquídeo, fue medida en 40 adultos normales a través de un ensayo inmunofluorométrico. El criterio diagnóstico estuvo basado en; muestras controles (pacientes normales) y muestras de pacientes con enfermedades que cursaron con disfunción de barrera sangre-líquido cefalorraquídeo. RESULTADOS: el coeficiente de correlación entre la lectina de unión a manosa en el líquido cefalorraquídeo y en el suero, fue muy bajo. El reibergrama de la lectina de unión a manosa se diseñó de acuerdo con procedimientos previos. CONCLUSIONES: bajo cualquier condición de barrera sangre-líquido cefalorraquídeo, el reibergrama puede identificar la ocurrencia de síntesis intratecal de lectina de unión a manosa.

BACKGROUND: The dynamics of brain derived proteins in cerebrospinal fluid is different from the dynamics of blood-derived proteins. Aim: To describe the empirical data for mannan binding lectin and gives a theoretical interpretation of the dynamics of this protein in cerebrospinal fluid through a new reibergram. METHODS: Serum and cerebrospinal fluid mannan binding lectin were measured in 40 normal adults by immunofluorometric assays. The diagnostic criteria were based in; normal control samples defined clinically and diseases with blood-cerebrospinal fluid barrier dysfunction. RESULTS: Correlation coefficient between cerebrospinal fluid MBL and serum MBL was very low. Mannan binding lectin reibergram was designed according with previous procedures. CONCLUSION: Under all conditions of the blood-cerebrospinal fluid barrier, the reibergram can identify the occurrence of intrathecal mannan binding lectin synthesis.

Bipolar and panic disorders may be associated with hereditary defects in the innate immune system.

BACKGROUND: Mannan-binding lectin (MBL) and mannan-binding lectin-associated serine protease-2 (MASP-2) represent important arms of the innate immune system, and different deficiencies may result in infections or autoimmune diseases. Both bipolar and panic disorders are associated with increased inflammatory response, infections and mutual comorbidity. However, associations with MBL, MASP-2 or the gene, MBL2, coding for MBL, have not been investigated thoroughly. METHODS: One hundred patients with bipolar disorder, 100 with panic disorder and 349 controls were included. Serum concentrations of MBL and MASP-2 were measured and seven single nucleotide polymorphisms (SNPs) influencing these concentrations were genotyped. Disease association with genetic markers and serum levels were investigated. RESULTS: In panic disorder, we observed a large proportion (30%) of MBL deficient (<100ng/ml) individuals and significantly lower levels of MBL and MASP-2 plus association with the MBL2 YA two-marker haplotype. Bipolar disorder was associated with the MBL2 LXPA haplotype and lower MASP-2 levels. LIMITATIONS: No information on course or severity of disorders was included, and only MBL and MASP-2 were measured, excluding other components from the complement pathway. Restrictions defined by ethnical committees preclude information of control׳s ethnic origin. CONCLUSIONS: Significant differences in MBL and MASP-2 concentrations were observed between cohorts, especially an intriguing finding associating panic disorder with MBL deficiency. These differences could not be fully explained by allele or haplotype frequency variations. Since MBL deficiency is highly heterogeneous and associated with both infectious and autoimmune states, more research is needed to identify which complement pathway components could be associated with bipolar respectively panic disorder.

Polymorphisms in the MASP1 gene are associated with serum levels of MASP-1, MASP-3, and MAp44.

INTRODUCTION: MASP-1 is the first protein in the activation of the lectin pathway and MASP-1 is, like its isoforms MASP-3 and MAp44, encoded by the MASP1 gene. Our aim was to explore associations between polymorphisms in MASP1 and corresponding concentrations of MASP-1, MASP-3, and MAp44 in plasma as well as the genetic contribution to the equilibrium between the three proteins. METHODS: Fifteen SNPs were genotyped in the MASP1 gene in 350 blood donors. Corresponding plasma concentrations of MASP-1, MASP-3, and MAp44 were measured. RESULTS: A total of 10 different SNPs showed associations with the concentration of one or some of the three proteins (rs113938200, rs190590338, rs35089177, rs3774275, rs67143992, rs698090, rs72549154, rs72549254, rs75284004, rs7625133), and several of these were in strong linkage. SNPs located in the mutually exclusive splice region had opposite effects on the protein concentrations. Being e.g. homozygote for the minor allele of rs3774275 was associated with an increase in median concentration of 13% in MASP-1(P=0.03), 29% in MAp44 (P<0.001), and a decrease in MASP-3 of 26% (P<0.001) compared to homozygosis for the major allele. Heterozygosis of rs113938200 (p.Asn368Asp in MAp44) was associated with a reduced MAp44 concentration of 61% (P=0.005). Rs190590338 located in the promoter region was associated in the heterozygote form with an increased MASP-1 concentration of 35% (P = 0.002). A multivariate linear regression model including sex, age, M- and H-ficolin, MBL, and the 15 SNPs explained 20-48% of the variation in the concentration of the three proteins and the SNPs investigated contributed with the most explanatory power (12-23%). DISCUSSION: The present study described 10 SNPs, which were associated with the concentration of one or some of the three proteins originating from the MASP1 gene and in a multivariate model it was shown that the SNPs contributed with the most explanatory power to the protein concentrations.

Leprosy association with low MASP-2 levels generated by MASP2 haplotypes and polymorphisms flanking MAp19 exon 5.

BACKGROUND: The gene MASP2 (mannan-binding lectin (MBL)-associated serine protease 2) encodes two proteins, MASP-2 and MAp19 (MBL-associated protein of 19 kDa), bound in plasma to MBL and ficolins. The binding of MBL/MASP-2 and ficolin/MASP-2 complexes to microorganisms activates the lectin pathway of complement and may increase the ingestion of intracellular pathogens such as Mycobacterium leprae. METHODS: We haplotyped 11 MASP2 polymorphisms with multiplex sequence-specific PCR in 219 Brazilian leprosy patients (131 lepromatous, 29 borderline, 21 tuberculoid, 14 undetermined, 24 unspecified), 405 healthy Brazilians and 291 Danish blood donors with previously determined MASP-2 and MAp19 levels. We also evaluated MASP-2 levels in further 46 leprosy patients and 69 Brazilian controls. RESULTS: Two polymorphisms flanking exon 5 of MASP2 were associated with a dominant effect on high MASP-2 levels and an additive effect on low MAp19 levels. Patients presented lower MASP-2 levels (P = 0.0012) than controls. The frequency of the p.126L variant, associated with low MASP-2 levels (below 200 ng/mL), was higher in the patients (P = 0.0002, OR = 4.92), as was the frequency of genotypes with p.126L (P = 0.00006, OR = 5.96). The *1C2-l [AG] haplotype, which harbors p.126L and the deficiency-causing p.439H variant, has a dominant effect on the susceptibility to the disease (P = 0.007, OR = 4.15). Genotypes composed of the *2B1-i and/or *2B2A-i haplotypes, both associated with intermediate MASP-2 levels (200-600 ng/mL), were found to be protective against the disease (P = 0.0014, OR = 0.6). Low MASP-2 levels (P = 0.022), as well as corresponding genotypes with *1C2-l and/or *2A2-l but without *1B1-h or *1B2-h, were more frequent in the lepromatous than in other patients (P = 0.008, OR = 8.8). CONCLUSIONS: In contrast with MBL, low MASP-2 levels increase the susceptibility to leprosy in general and to lepromatous leprosy in particular. MASP2 genotypes and MASP-2 levels might thus be of prognostic value for leprosy progression.

Non-synonymous polymorphisms in the FCN1 gene determine ligand-binding ability and serum levels of M-ficolin.

BACKGROUND: The innate immune system encompasses various recognition molecules able to sense both exogenous and endogenous danger signals arising from pathogens or damaged host cells. One such pattern-recognition molecule is M-ficolin, which is capable of activating the complement system through the lectin pathway. The lectin pathway is multifaceted with activities spanning from complement activation to coagulation, autoimmunity, ischemia-reperfusion injury and embryogenesis. Our aim was to explore associations between SNPs in FCN1, encoding M-ficolin and corresponding protein concentrations, and the impact of non-synonymous SNPs on protein function. PRINCIPAL FINDINGS: We genotyped 26 polymorphisms in the FCN1 gene and found 8 of these to be associated with M-ficolin levels in a cohort of 346 blood donors. Four of those polymorphisms were located in the promoter region and exon 1 and were in high linkage disequilibrium (r(2)≥0.91). The most significant of those were the AA genotype of -144C>A (rs10117466), which was associated with an increase in M-ficolin concentration of 26% compared to the CC genotype. We created recombinant proteins corresponding to the five non-synonymous mutations encountered and found that the Ser268Pro (rs150625869) mutation lead to loss of M-ficolin production. This was backed up by clinical observations, indicating that an individual homozygote of Ser268Pro would be completely M-ficolin deficient. Furthermore, the Ala218Thr (rs148649884) and Asn289Ser (rs138055828) were both associated with low M-ficolin levels, and the mutations crippled the ligand-binding capability of the recombinant M-ficolin, as indicated by the low binding to Group B Streptococcus. SIGNIFICANCE: Overall, our study interlinks the genotype and phenotype relationship concerning polymorphisms in FCN1 and corresponding concentrations and biological functions of M-ficolin. The elucidations of these associations provide information for future genetic studies in the lectin pathway and complement system.

Mannan-binding lectin in cerebrospinal fluid: a leptomeningeal protein.

BACKGROUND: Mannan-binding lectin (MBL), a protein of the innate immune response is attracting increasing clinical interest, in particularly in relation to its deficiency. Due to its involvement in brain diseases, identifying the source of MBL in CSF is important. Analysis of cerebrospinal fluid (CSF) can provide data that discriminates between blood-, brain-, and leptomeninges-derived proteins. To detect the source of MBL in CSF we need to consider three variables: the molecular size-dependent concentration gradient between CSF and blood, the variation in transfer between blood and CSF, and the CSF MBL concentration correlation with the albumin CSF/serum quotient (QAlb), i.e., with CSF flow rate. METHODS: MBL was assayed in samples of CSF and serum with an ELISA, coated with anti MBL antibodies. Routine parameters such as albumin-, immunoglobulin- CSF/serum quotients, oligoclonal IgG and cell count were used to characterize the patient groups. Groups comprised firstly, control patients without organic brain disease with normal CSF and normal barrier function and secondly, patients without inflammatory diseases but with increased QAlb, i.e. with a blood CSF barrier dysfunction. RESULTS: MBL concentration in CSF was at least five-fold higher than expected for a molecular-size-dependent passage from blood. Secondly, in a QIgM/QAlb quotient diagram (Reibergram) 9/13 cases showed an intrathecal fraction in some cases over 80% of total CSF MBL concentration 3) The smaller inter-individual variation of MBL concentrations in CSF of the control group (CV = 66%) compared to the MBL concentrations in serum (CV = 146%) indicate an independent source of MBL in CSF. 4) The absolute MBL concentration in CSF increases with increasing QAlb. Among brain-derived proteins in CSF only the leptomeningeal proteins showed a (linear) increase with decreasing CSF flow rate, neuronal and glial proteins are invariant to changes of QAlb. CONCLUSIONS: MBL in CSF is predominantly brain-derived and all results pointed to the leptomeningeal cells as the source of the protein. The evaluation of this protein requires the interpretation of its absolute concentrations in CSF as a function of the albumin quotient, QAlb. This recognition of MBL in brain cells opens a new field of discussion about the function of the innate immune response in CNS in cases of acute and chronic neurological diseases.