M-ficolin Levels Research Articles

Complement Proteins L-Ficolin and M-Ficolin Are Increased in Patients With Axial Spondyloarthritis and Decrease After Tumor Necrosis Factor Inhibitor Treatment.

We have previously reported elevated levels of the complement lectin pathway proteins L-ficolin and H-ficolin in patients with axial spondyloarthritis (axSpA) compared with healthy controls. The aim of the present study was to investigate these biomarkers in a cross-sectional cohort of patients suffering from low back pain (LBP). Further, we aimed to investigate changes in lectin pathway protein levels after initiation of adalimumab (ADA; a tumor necrosis factor inhibitor) in a longitudinal cohort of patients with axSpA. Lectin pathway protein levels (mannan-binding lectin [MBL], collectin liver 1, H-ficolin, L-ficolin, M-ficolin, MBL-associated serine protease [MASP]-1, MASP-2, MASP-3, MBL-associated protein 19 [MAp19], and MAp44) in EDTA plasma were determined in 2 well-characterized cohorts: (1) a clinical cross-sectional cohort of patients with LBP, including patients with axSpA (n = 23), patients with unspecific LBP (uLBP) with ≥ 1 SpA features (n = 55), and patients with uLBP without SpA features or magnetic resonance imaging findings suggestive of axSpA (n = 64); and (2) a randomized double-blinded, placebo-controlled trial cohort of patients with axSpA (n = 49) initiating ADA therapy. Lectin pathway protein levels were determined using immunoassays. Plasma levels of L-ficolin and M-ficolin were significantly increased in the cross-sectional cohort of newly diagnosed patients with axSpA compared with clinically relevant controls with uLBP (all P < 0.05). Both L-ficolin and M-ficolin decreased significantly after ADA therapy (P < 0.05). L-ficolin and M-ficolin levels are elevated in newly diagnosed patients with axSpA compared with clinically relevant controls. Both L-ficolin and M-ficolin levels decrease significantly after initiating ADA therapy. These findings provide new insights into the inflammatory processes in axSpA and support the involvement of complement in axSpA pathogenesis.

POS0059 COMPLEMENT PROTEINS ARE ELEVATED IN PATIENTS WITH axSpA COMPARED WITH RELEVANT CONTROLS OF PATIENTS WITH LOW BACK PAIN AND SpA-FEATURES WITHOUT AXSPA.

BackgroundAxial spondyloarthritis (axSpA) is associated with a certain genetic predisposition, i.e., with the presence of human leukocyte antigen (HLA)-B27. However, the pathogenesis remains largely unexplained. Animal models of ankylosing spondylitis have shown inhibition of complement to be beneficial in terms of limiting structural damage (1). The lectin pathway of complement activation serves as a key component of the innate immune system and plays a pivotal role in both homeostasis and development. The influence of complement in axSpA is mainly unexplored. We have, however, previously reported elevated plasma levels of the lectin pathway proteins L-ficolin and H-ficolin in patients with axSpA compared with blood donors (2).ObjectivesOur aim was to investigate plasma levels of lectin pathway proteins in a clinical cohort of patients with axSpA and compare them to relevant controls that we often experience significant challenges in differentiating from axSpA.MethodsPlasma samples were obtained from individuals in a cohort of patients suffering from low back pain (LBP) including: 1) 23 patients with axSpA, 2) 55 patients without axSpA experiencing SpA-features/symptoms, and 3) 64 patients with nonspecific LBP without SpA-features or MRI findings suggestive of axSpA. Diagnosis of axSpA was based on multidisciplinary team conference consensus after 3.5 years of follow-up (3). Plasma levels of 10 lectin pathway proteins (MBL, CL-L1, H-ficolin, L-ficolin, M-ficolin, MASP-1, MASP-2, MASP-3, MAp44, and MAp19) were measured by immunoassays developed in-house.ResultsPatient characteristics are shown in Table 1. Plasma levels of lectin pathway proteins L-ficolin, M-ficolin and CL-L1 differed significantly in the patient groups (p ≤ 0.03). L-ficolin and M-ficolin were elevated in axSpA-patients compared with patients with SpA-features without axSpA and nonspecific LBP patients (Figure 1). CL-L1 was elevated in axSpA-patients and patients with SpA-features without axSpA compared with nonspecific LBP patients (Figure 1). No significant differences were observed for MBL, H-ficolin, MASP-1, MASP-2, MASP-3, MAp44, and MAp19. L-ficolin levels correlated with CRP in axSpA-patients (Spearman’s rho=0.58 p=0.004). M-ficolin levels correlated weakly with CRP in nonspecific LBP patients (Spearman’s rho=0.36 p=0.003). Lectin pathway protein levels did not correlate with disease activity (ASDAS).Table 1.Patient characteristicsaxSpA (n=23)Not axSpA (n=55)Non-specific low back pain (n=64)p-valueMedian age, years (range)32 (19-40)33 (19-41)32 (18-39)0.75aMales, n (%)10 (43)37 (67)26 (41)0.01bHLA-B27 positive, n (%)17 (74)11 (20)5 (8)0.00bInflammatory back pain, n (%)18 (78)28 (51)0.03cGood response to NSAID14 (61)17 (31)0.01cSacroiliitiis on MRI acc. ASAS, n (%)22 (96)45 (82)0.11cElevated CRP, n (%)3 (13)7 (13)0.97ASDAS (range)2.5 (1.2-3.7)2.3 (0.8-3.8)0.52da compared by Kruskal-Wallis test.b all three groups compared by Chi2 test.c compared by Chi2 test.d compared by Mann Whitney U test.Figure 1.Plasma levels of L-ficolin, M-ficolin and CL-L1.ConclusionL-ficolin and M-ficolin are increased in patients with axSpA when compared with relevant control cohorts of patients with LBP or with SpA-features without axSpA. Our findings support a potential pathogenic role for complement in axSpA, however, further studies are needed to elucidate the diagnostic potential of the specific complement proteins.

Pattern Recognition Molecules of Lectin Complement Pathway in Ischemic Stroke.

PurposeThe 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 MethodsA 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.ResultsAccording 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.ConclusionIn 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.

Remote ischemic preconditioning does not influence lectin pathway protein levels in head and neck cancer patients undergoing surgery.

Cancer patients who undergo tumor removal, and reconstructive surgery by transfer of a free tissue flap, are at high risk of surgical site infection and ischemia-reperfusion injury. Complement activation through the lectin pathway (LP) may contribute to ischemia-reperfusion injury. Remote ischemic preconditioning (RIPC) is a recent experimental treatment targeting ischemia-reperfusion injury. The study aims were to investigate LP protein plasma levels in head and neck cancer patients compared with healthy individuals, to explore whether RIPC affects LP protein levels in head and neck cancer surgery, and finally to examine the association between postoperative LP protein levels and the risk of surgical site infection. Head and neck cancer patients (n = 60) undergoing tumor resection and reconstructive surgery were randomized 1:1 to RIPC or sham intervention administered intraoperatively. Blood samples were obtained preoperatively, 6 hours after RIPC/sham, and on the first postoperative day. LP protein plasma levels were measured utilizing time-resolved immunofluorometric assays. H-ficolin and M-ficolin levels were significantly increased in cancer patients compared with healthy individuals (both P ≤ 0.02). Conversely, mannan-binding lectin (MBL)-associated serine protease (MASP)-1, MASP-3, collectin liver-1 (CL-L1), and MBL-associated protein of 44 kilodalton (MAp44) levels were decreased in cancer patients compared with healthy individuals (all P ≤ 0.04). A significant reduction in all LP protein levels was observed after surgery (all P < 0.001); however, RIPC did not affect LP protein levels. No difference was demonstrated in postoperative LP protein levels between patients who developed surgical site infection and patients who did not (all P > 0.13). The LP was altered in head and neck cancer patients. LP protein levels were reduced after surgery, but intraoperative RIPC did not influence the LP. Postoperative LP protein levels were not associated with surgical site infection.

Ficolin‐1 gene (FCN1) −144 C/A polymorphism is associated with adverse outcome of severe pneumonia in the under‐five Egyptian children: A multicenter study

Pneumonia is the foremost cause of child death worldwide. M-ficolin is encoded by the FCN1 gene and represents a novel link between innate and adaptive immunity. To investigate the FCN1 -144 C/A (rs10117466) polymorphism as a potential marker for pneumonia severity and adverse outcome namely complications or mortality in the under-five Egyptian children. This was a prospective multicenter study that included 620 children hospitalized with World Health Organization-defined severe pneumonia and 620 matched healthy control children. Polymorphism rs10117466 of the FCN1 gene promoter was analyzed by PCR-SSP, while serum M-ficolin levels were assessed by ELISA. The FCN1 A/A genotype and A allele at the -144 position were more frequently observed in patients compared to the control children (43.4% vs 27.6%; odds ratio [OR]: 1.62; [95% confidence interval {CI}: 1.18-2.2]; for the A/A genotype) and (60.8% vs 52.5%; OR: 1.4; [95% CI: 1.19-1.65]; for the A allele); P < .01. The FCN1 -144 A/A homozygous patients had significantly higher serum M-ficolin concentrations (mean: 1844 ± 396 ng/mL) compared with those carrying the C/C or C/A genotype (mean: 857 ± 278 and 1073 ± 323 ng/mL, respectively; P = .002). FCN1 -144 A/A genotype was an independent risk factor for adverse outcomes in children with severe pneumonia (adjusted OR = 4.85, [95% CI: 2.96-10.25]; P = .01). The FCN1 A/A genotype at the -144 position was associated with high M-ficolin serum levels and possibly contributes to enhanced inflammatory response resulting in the adverse outcome of pneumonia in the under-five Egyptian children.

Complement lectin pathway protein levels reflect disease activity in juvenile idiopathic arthritis: a longitudinal study of the Nordic JIA cohort

BackgroundTo determine the serum levels of the lectin pathway proteins early in the disease course and 17 years after disease onset and to correlate the protein levels to markers of disease activity in participants from a population-based Nordic juvenile idiopathic arthritis (JIA) cohort. Additionally, to assess the predictive value of lectin pathway proteins with respect to remission status.MethodsA population-based cohort study of consecutive cases of JIA with a disease onset from 1997 to 2000 from defined geographical areas of Finland, Sweden, Norway and Denmark with 17 years of follow-up was performed. Clinical characteristics were registered and H-ficolin, M-ficolin, MASP-1, MASP-3, MBL and CL-K1 levels in serum were analyzed.ResultsIn total, 293 patients with JIA were included (mean age 23.7 ± 4.4 years; mean follow-up 17.2 ± 1.7 years). Concentrations of the lectin protein levels in serum were higher at baseline compared to the levels 17 years after disease onset (p ≤ 0.006, n = 164). At baseline, the highest level of M-ficolin was observed in systemic JIA. Further, high M-ficolin levels at baseline and at 17-year follow-up were correlated to high levels of ESR. In contrast, high MASP-1 and MASP-3 tended to correlate to low ESR. CL-K1 showed a negative correlation to JADAS71 at baseline.None of the protein levels had prognostic abilities for remission status 17 years after disease onset.ConclusionWe hypothesize that increased serum M-ficolin levels are associated with higher disease activity in JIA and further, the results indicate that MASP-1, MASP-3 and CL-K1 are markers of inflammation.

The complement lectin pathway after cardiac arrest.

The lectin pathway (LP) of the complement system may initiate inflammatory reactions when body tissue is altered. We aimed to investigate the levels of the LP proteins in out-of-hospital cardiac arrest patients, and to compare these with healthy individuals. Furthermore, we aimed to clarify whether the duration of targeted temperature management influenced LP protein levels, and we further examined whether LP proteins were associated with 30-day mortality. We included 82 patients resuscitated from out-of-hospital cardiac arrest. The patients were randomly assigned to 24 or 48hours of targeted temperature management at 33±1°C. Blood samples were obtained 22, 46 and 70hours after target temperature was reached. Levels of the LP proteins (mannan-binding lectin [MBL], M-ficolin, H-ficolin, collectin liver 1 [CL-L1], MBL-associated serine protease 1 [MASP-1], MASP-2, MASP-3 and MBL-associated protein of 44kDa [MAp44]) were measured using time-resolved immunofluorometric assays. Data from 82 gender matched healthy individuals were used for comparison. Levels of CL-L1, MASP-1, MASP-2 and MAp44 were significantly higher, whereas M-ficolin levels were significantly lower in cardiac arrest patients compared with healthy individuals. MASP-2, MASP-3 and M-ficolin levels changed significantly when comparing 24 and 48 hours of targeted temperature management. The LP protein levels were not different between 30-day survivors and non-survivors after cardiac arrest. The differences in LP protein levels between patients and healthy individuals may indicate that cardiac arrest patients have an activated LP. Overall, the LP protein levels were not influenced by the duration of targeted temperature management, and the levels were not associated with 30-day mortality.

The lectin pathway of the complement system is downregulated in Crohn's disease patients who respond to anti-TNF-α therapy

Background and aimsThe lectin pathway of the complement system is initiated through the recognition of pathogens or altered self-structures by mannan-binding lectin (MBL) or ficolins and subsequent activation of MBL-associated serine proteases (MASPs). Altered ficolin levels may contribute to a dysregulated immune response in Crohn's disease (CD). A complete analysis of the lectin pathway has not been performed in patients with CD. We hypothesised that the lectin pathway proteins exacerbate inflammation in CD. MethodsWe assessed the lectin pathway proteins in 43 patients with active CD & 350 blood donors by measuring the serum levels of MBL; M-, H-, and L-ficolin; MASP-2; MASP-3; and MAp44. In patients with CD, the blood samples were obtained during induction treatment with infliximab or adalimumab. ResultsOf 43 patients with CD, 32 (74%) were classified as responders. We observed a nearly 50% decrease in median M-ficolin levels between day 0 and weeks 1/7 in the responders (p<0.001), whereas there was no decrease in the non-responders. MASP-2 levels decreased from baseline to week 1 in both the responders (37%, p<0.0001) and the non-responders (29%, p=0.02). In the responders only, the level of the inhibitory serine protease MASP-3 increased by 26% from baseline to week 1 (p<0.001) and remained high at week 7. ConclusionsOur findings indicated that M-ficolin, MASP-2, and MASP-3 may act in concert to reduce the activity of the lectin pathway, in patients with CD who respond to biological therapy.

OP0223 M-ficolin, an activator of the complement system, predicts DAS28 remission in early DMARD naïve rheumatoid arthritis

BackgroundM-ficolin is a soluble pattern recognition molecule that activates the complement system. We recently reported a factor 300 difference in the synovial fluid M-ficolin concentration between rheumatoid arthritis (RA) and...

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

BackgroundThe 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 FindingsWe 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 (r2≥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.SignificanceOverall, 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.

Susceptibility to Leprosy is Associated with M-ficolin Polymorphisms

Mycobacterium leprae exploits complement activation and opsonophagocytosis to infect phagocytes. M-ficolin is encoded by the FCN1 gene and initiates the lectin pathway on monocyte surfaces. We investigated FCN1 promoter polymorphisms that could be responsible for the high interindividual variability of M-ficolin levels and for modulating leprosy susceptibility. We genotyped rs2989727 (-1981 G > A), rs28909068 (-791 G > A), rs10120023 (-542 G > A), rs17039495 (-399 G > A), rs28909976 (-271IndelT), rs10117466 (-144C > A) and rs10858293 (+33 T > G) in 400 controls and 315 leprosy patients from Southern Brazil, and in 296 Danish healthy individuals with known M-ficolin levels. Ten haplotypes were identified with sequence-specific PCR and/or haplotype-specific sequencing. We found evidence for a protective codominant additive effect of FCN1*-542A-144C with leprosy in Euro-Brazilians (P=0.003, PBf =0.021, OR=0.243 [CI95% =0.083-0.71]), which was independent of age, ethnic group and gender effects (P=0.029). There was a trend for a positive association of the -399A variant in Afro-Brazilians (P=0.022, PBf =0.154, OR=4.151 [CI95% =1.115-15.454], as well as for a negative association of the FCN1*3A haplotype with lepromatous leprosy, compared with less severe forms of the disease (P=0.016, PBf =0.112, OR=0.324 [CI95% =0.123-0.858]). Danish individuals with this haplotype presented M-ficolin levels higher than the population average of circa 1,000 ng/ml, and -542A-144C, which is able to modify the recognition of transcription factors in silico, occurred in individuals with levels under the 25 percentil (P=0.031). Our data provide the first evidence that FCN1 polymorphisms are associated with leprosy. M-ficolin may represent a novel key to understand the immunopathogenesis of M. leprae infection.

M-ficolin levels are associated with the occurrence of severe infections in patients with haematological cancer undergoing chemotherapy

The pattern recognition molecules H-ficolin, L-ficolin and M-ficolin bind to micro-organisms. They activate the lectin pathway of complement through mannan-binding lectin (MBL)-associated serine proteases (MASPs). Association between low MBL levels and infections in patients undergoing chemotherapy for haematological diseases has been observed previously. We now examine for MASP-2, MASP-3 and ficolin levels. We assessed the concentration of lectin pathway molecules as risk factors for infection in patients with haematological malignancy undergoing chemotherapy. Samples taken before the initiation of chemotherapy covering 117 chemotherapy cycles in 105 patients were available. MASPs and ficolins were measured by time-resolved immunoflourometric assays and the levels related to parameters of infections. End-points included febrile neutropenia, documented infections, bacteraemia or severe infections. Lower M-ficolin concentrations were found in patients who developed a severe infection: median 0·27 µg/ml compared to 0·47 µg/ml in patients who did not develop a severe infection (P = 0·01). Conversely, MASP-2 was higher in these patients: median 0·53 µg/ml compared to 0·37 µg/ml, respectively (P = 0·008). When considering M-ficolin levels below 0·36 µg/ml as deficient, the time to development of severe infection was shorter in the M-ficolin deficient group: the hazard ratio was 2·60 (95% confidence interval: 1·23-5·49). No associations were revealed between infections and H-ficolin, L-ficolin or MASP-3. Patients with low M-ficolin are more likely to develop severe infections, whereas MASP-2 showed the opposite.

Levels of lectin pathway proteins in plasma and synovial fluid of rheumatoid arthritis and osteoarthritis

The innate immune system contributes to the development of rheumatoid arthritis (RA). A potent contributor to such processes is the complement system. The complement system is known to be activated in the inflammatory phases of osteoarthritis (OA). The lectin pathway of the complement system is activated through the recognition of pathogens or altered self-structures by mannan-binding lectin (MBL) or one of the three ficolins in collaboration with MBL-associated serine proteases (MASPs). We assessed the lectin pathway in plasma and synovial fluid (SF) of 27 RA patients and 30 OA patients by measuring MBL, MASP-2, MASP-3, M-ficolin, and H-ficolin. The concentration for all 5 proteins was significantly higher in plasma than in SF (P < 0.001) and the concentration in paired plasma and SF samples correlated in both RA and OA (significance levels between <0.001 and 0.02). The ratio of SF/plasma concentration was for all proteins significantly elevated in RA compared with OA patients (all P < 0.001). The M-ficolin concentration correlated with the neutrophils in both plasma (P = 0.01) and SF (P < 0.001) of RA, and in plasma of 78 controls (P = 0.03). To our knowledge, this is the first report on these proteins in SF, except for MBL where our results are in contrast to the one previous publication. The results support an important physiological role of the neutrophils in determining the M-ficolin levels in both RA and healthy adults. We suggest that quantifications of white blood cells should be included in future clinical investigations of M-ficolin.

Characteristics and Biological Variations of M-Ficolin, a Pattern Recognition Molecule, in Plasma

The three human ficolins, H-ficolin, L-ficolin and M-ficolin, are pattern recognition molecules of the innate immune system. All three ficolins can activate the lectin pathway of the complement system after binding to pathogens. H- and L-ficolin are serum proteins with an average concentration of 18 and 3 µg/ml, respectively. M-ficolin has been described as a membrane-associated pattern recognition receptor of monocytes, being also present in granulocytes; recently, minuscule amounts of M-ficolin have been found in serum, too. No assay specific for M-ficolin has yet been described and biological variations are unknown. We have raised specific monoclonal anti-human M-ficolin antibodies and have developed a quantitative assay for M-ficolin. M-ficolin elutes as a large, 900-kDa protein upon gel permeation chromatography of serum. Analysis of M-ficolin levels in serum samples of 350 blood donors reveals a mean concentration of 1.07 µg/ml, ranging from 0.28 to 4.05 µg/ml. Analyses of consecutive acute phase serum samples from major surgery patients indicated a complex response. Ontogeny was investigated through cord blood samples from healthy full-term babies, which showed adult levels, with sequential samples showing no increase from birth to 1 year of age. We suggest that M-ficolin should also be considered as a humoral pattern recognition molecule.