Ficolin-2 and ficolin-3 in women with malignant and benign ovarian tumours.

Ficolins are serum pattern recognition molecules. They have opsonic properties and are able to activate complement via the lectin pathway. This paper reports investigations concerning ficolin-2 and ficolin-3 in ovarian cancer (OC). Their serum levels, single nucleotide polymorphisms of the corresponding FCN2 and FCN3 genes and specific mRNA expression in ovarian sections were investigated in 128 patients suffering from primary OC and 197 controls operated on for reasons other than malignancies. The latter consisted of two reference groups: those with benign tumours (n = 123) and those with normal ovaries (NO) (n = 74). Serum ficolin-2 and ficolin-3 concentrations were higher among patients with malignant disease when compared with either of the reference groups. A significant correlation between ficolin-2 and ficolin-3 concentrations was found, while no correlations with CA125 antigen or CRP were observed. No differences in the frequency of single nucleotide polymorphisms at sites -64, -4 (promoter), +6359, or +6424 (exon 8) (FCN2 gene) nor in the frame-shift mutation 1637delC (FCN3 gene) were found between investigated groups. In contrast to serum concentrations, the expression of FCN2 gene (reported for the first time in ovarian sections) was significantly lower in women with OC in comparison with patients with NO but not with benign ovarian tumours. In case of FCN3 gene, its expression levels in OC group inversely correlated with serum ficolin-3 and were lower in comparison with controls.

Human L-ficolin (ficolin-2) and its clinical significance.

Human L-ficolin (P35, ficolin-2) is synthesised in the liver and secreted into the bloodstream where it is one of the major pattern recognition molecules of plasma/serum. Like other ficolins, it consists of a collagen-like tail region linked to a fibrinogen-related globular head; a basic triplet subunit arises via a collagen-like triple helix, and this then forms higher multimers (typically a 12-mer, Mr 400K). Unlike other ficolins, it has a complex set of binding sites arranged within an internal cleft enabling it to recognise a variety of molecular patterns including acetylated sugars and certain 1,3-ß-glucans. It is one of the few molecules known to activate the lectin pathway of complement. Recently, some disease association studies (at either the DNA or protein level) have implicated L-ficolin in innate immunity, where it might cooperate with pentraxins and collectins. Emerging lines of evidence point to a role for L-ficolin in respiratory immunity, where its affinity for Pseudomonas aeruginosa could be significant.

Factors of the lectin pathway of complement activation and their clinical associations in neonates.

This paper summarizes the data concerning soluble defense lectins (mannan-binding lectin, M-ficolin, L-ficolin, and H-ficolin) with the unique ability to activate complement and their associated serine proteases (MASPs) in neonates. The clinical importance of deficiencies of these immune factors is presented in aspects of perinatal mortality, premature births, and low birthweight. Prenatal serum concentrations of L-ficolin, H-ficolin, and MASP-2 (and probably M-ficolin) correlate with gestational age and birthweight. The relationship of serum MBL to gestational age is controversial. The MBL2 genotypes XA/O and O/O (associated with low-serum MBL) are associated with perinatal infections, whereas the high serum MBL-conferring A/A genotypes may be associated with prematurity. Low-serum L-ficolin concentrations, but not low-serum H-ficolin concentrations, are also associated with perinatal infections. Much of the literature is inconsistent, and the relationships reported so far require independent confirmation at both gene and protein levels. Our preliminary conclusion is that these soluble defense lectins play a protective role in the neonate, and that insufficiency of such factors contributes to the adverse consequences of prematurity and low birthweight.

Impact of mannose-binding lectin insufficiency on the course of cystic fibrosis: A review and meta-analysis.

Mannose-binding lectin (MBL) is an innate immune protein produced by the liver. MBL binds to glycoconjugates containing mannose, fucose or N-acetylglucosamine that are present in a wide variety of bacteria, viruses and fungi. Upon binding, MBL may active the lectin pathway of complement or directly opsonize organisms to enhance phagocytosis. MBL is primarily a serum protein but accumulates in the lung during acute inflammation. Recent evidence suggests an important role for MBL in a variety of infectious disorders. Cystic fibrosis (CF) is a multisystem disease caused by mutations in the gene encoding the CF transmembrane regulator (CFTR). The course of CF lung disease is highly variable even in patients with the same CFTR genotype, suggesting that other modulator genes are important for prognosis. MBL has been proposed as a possible modulator of clinical severity in CF. In this review and meta-analysis, we found that MBL2 genotypes associated with MBL insufficiency were associated with earlier acquisition of Pseudomonas aeruginosa (P < 0.0001), reduced pulmonary function among adult patients (P < 0.0001 for forced expiratory volume), and an increased rate of death or requirement for lung transplantation (odds ratio 3.69; P = 0.02). The available evidence therefore suggests that MBL insufficiency is associated with the severity of CF lung disease. The possible future prophylactic or therapeutic application of MBL replacement is discussed.

Polymorphisms of the FCN2 Gene 3'UTR Region and Their Clinical Associations in Preterm Newborns.

Ficolin-2 is regarded as an important innate immunity factor endowed with both lectin (carbohydrate recognition) qualities and ability to induce complement activation. The aim of this study was to investigate the association of the FCN2 3'-untranslated region (3'UTR) polymorphisms with ficolin-2 expression and perinatal complications in preterm neonates. The sequencing analysis allowed us to identify six 3'UTR polymorphisms with minor allele frequency (MAF) >1%: rs4521835, rs73664188, rs11103564, rs11103565, rs6537958 and rs6537959. Except for rs4521835, all adhered to Hardy-Weinberg expectations. Moreover, rs6537958 and rs6537959 were shown to be in perfect linkage disequilibrium (LD) with nine other genetic polymorphisms: rs7040372, rs7046516, rs747422, rs7847431, rs6537957, rs6537960, rs6537962, rs11462298 and rs7860507 together stretched on a distance of 1242 bp and very high LD with rs11103565. The 3'UTR region was shown to bind nuclear extract proteins. The polymorphisms at rs4521835 and rs73664188 were found to influence serum ficolin-2 concentration significantly. All polymorphisms identified create (together with exon 8 polymorphism, rs7851696) two haplotype blocks. Among 49 diplotypes (D1-D49) created from rs7851696 (G>T), rs4521835 (T>G), rs73664188 (T>C), rs11103564 (T>C), rs11103565 (G>A) and rs6537959 (T>A), twenty two occurred with frequency >1%. Two diplotypes: D13 (GTTTGT/GGTCGT) and D10 (GTTTGT/GGTCGA), were significantly more frequent among preterm neonates with early onset of infection and pneumonia, compared with newborns with no infectious complications (OR 2.69 and 2.81, respectively; both p<0.05). The minor (C) allele at rs73664188 was associated with an increased risk of very low (≤1500 g) birthweight (OR=1.95, p=0.042) but was associated with the opposite effect at rs11103564 (OR=0.11, p=0.005).

Two factors of the lectin pathway of complement, l-ficolin and mannan-binding lectin, and their associations with prematurity, low birthweight and infections in a large cohort of Polish neonates.

Ficolins and one collectin, mannan-binding lectin (MBL), are the only factors known to activate the lectin pathway (LP) of complement. There is considerable circumstantial evidence that MBL insufficiency can increase susceptibility to various infections and influence the course of several non-infectious diseases complicated by infections. Much less information is available concerning l-ficolin. We report the results of a prospective study to investigate any association between either MBL deficiency or l-ficolin deficiency with prematurity, low birthweight or perinatal infections in a large cohort of Polish neonates, representing an ethnically homogenous population (n=1832). Cord blood samples were analysed to determine mbl-2 gene variants, MBL concentrations and MBL-MASP-2 complex activities (MBL-dependent lectin pathway activity) as well as l-ficolin levels. Median concentrations of l-ficolin and MBL were 2500 and 1124 ng/ml, respectively, while median LP activity was 272 mU/ml. After genotyping, 60.6% of babies were mbl-2 A/A, 35.4% were A/O and 4% were O/O genotypes. We found relative l-ficolin deficiency to be associated with prematurity, low birthweight and infections. l-Ficolin concentration correlated with gestational age and with birthweight, independently of gestational age. Preterm deliveries (<38 weeks) occurred more frequently among neonates with low LP activity but not with those having low serum MBL levels. Similarly, no association of serum MBL deficiency with low birthweight was found, but there was a correlation between LP activity and birthweight. Genotypes conferring very low serum MBL concentrations were associated with perinatal infections, and high-MBL-conferring genotypes were associated with prematurity. Our findings suggest that l-ficolin participates in host defence during the perinatal period and constitute the first evidence that relative l-ficolin deficiency may contribute to the adverse consequences of prematurity. Some similar trends were found with facets of MBL deficiency, but the observed relationships were weaker and less consistent.

Mannan-binding lectin genotypes and genotype-phenotype relationships in a large cohort of Polish neonates.

Circulating mannan (or mannose)-binding lectin (MBL) is genetically determined. Low MBL concentrations are associated with certain point mutations in the human MBL2 gene. Here we report the full MBL2 genotypes of 1800 Polish neonates and relate individual genotypes to serum MBL and MBL-dependent activity of the lectin pathway of complement activation. The seven acknowledged common haplotypes were found, plus the uncommon LYPD haplotype, combining to form 33 genotypes in this population. As expected, a strong correlation existed between genotypes and serum MBL or lectin pathway activity, and the latter two entities correlated strongly with each other. However, serum MBL values varied up to greater than 90-fold within genotypes. Unexpectedly, higher lectin pathway activity was found in association with the P allele relative to the Q allele. These data from a large cohort of neonates, representing an ethnically homogenous population, suggest that the current knowledge of the genetics of MBL2 is inadequate to predict serum MBL concentration and MBL-dependent lectin pathway activity in individual subjects.

Lectin-glycoconjugate interactions in health and disease.

It is increasingly being acknowledged that complex carbohydrates mediate a huge variety of cellular interactions, permitting and regulating recognition and signalling events. This is achieved by the enormous range and complexity of branched structures in glycoconjugates and the ability of carbohydrate-binding proteins (lectins) to decipher this 'glycocode'. Approx. 120 participants attended the 23rd International Lectin Meeting (Interlec-23) held at the Universities of Edinburgh (2 days) and Stirling (4 days) between 11 and 16 July 2008. These 'Interlecs' are truly international multi-disciplinary symposia, providing opportunities for scientists from different backgrounds, but with a common interest in some aspect of protein-carbohydrate interactions, to present their work in an informal and stimulating atmosphere. A major aim is always to induce cross-fertilization of ideas and concepts, and Interlec-23 was intended to have some bias towards lectins (galectins, collectins, selectins, siglecs etc.) and their ligands in human health and disease. Delegates from over 30 countries attended this meeting which was divided into seven oral sessions opened by a keynote speaker. This issue of Biochemical Society Transactions contains papers based on the keynote lectures and is therefore representative of the main themes of Interlec-23.

Is mannan-binding lectin (MBL) detectable on monocytes and monocyte-derived immature dendritic cells?

MBL (mannan-binding lectin; also called mannose-binding lectin) is a circulating C-type lectin with a collagen-like region synthesized mainly by the liver. MBL may influence susceptibility to infection in recipients of stem cell transplants, and it has even been suggested that the MBL status of a donor can influence the recipient's susceptibility to post-transplant infections. We have previously reported that MBL can be detected on human monocytes and monocyte-derived dendritic cells, based on detection using biotinylated anti-MBL, suggesting that those cells could synthesize MBL. If true, permanent MBL replacement therapy could be achieved by stem cell infusions. However, two other groups independently failed to find mbl-2-derived mRNA in monocytes. Therefore, to confirm or refute our previous observations, we used an alternative experimental strategy. Instead of using biotinylated antibody and labelled streptavidin, detection of surface MBL was attempted using MBL-specific primary antibodies (131-1, 131-10 and 131-11) followed by fluorescein-labelled anti-IgG, and controlled by the use of non-specific IgG as primary antibody. Monocytes were counterstained with anti-CD14-PE before FACS analysis. Adherent monocytes were also cultured for 48 h in serum-free medium or converted into immature dendritic cells by culture with IL-4 (interleukin-4) and GM-CSF (granulocyte/monocyte colony-stimulating factor). During FACS analysis, the dendritic cells were gated after counter-staining with anti-CD1a-PE. MBL was readily detected on the surface of fresh monocytes using all three specific anti-MBL monoclonal antibodies, but specific anti-MBL binding was greatly diminished after monocytes had been cultured for 2 days in serum-free medium. Moreover, we could not detect any MBL present on the surface of monocyte-derived dendritic cells. We therefore conclude that MBL is indeed present on the surface of fresh human monocytes. However, in view of the mRNA findings of others and our own previous observation that no secretion of MBL took place in culture, we presume that the surface-bound MBL is derived from autologous plasma and not synthesized by the cells. This conclusion is consistent with our in vivo findings in stem cell transplant patients which provided evidence against significant extra-hepatic production of serum MBL. It provides no ready explanation for the remarkable observation of Mullighan, Heatley, Doherty, Szabo, Grigg, Hughes, Schwarer, Szer, Tait, Bik To and Bardy [(2002) Blood 99, 3524-3529] that the presence of variant alleles of mbl-2 in stem cell donors can influence susceptibility to serious infections in their recipients.

Components of the lectin pathway of complement activation in paediatric patients of intensive care units.

Infections are a major cause of childhood mortality. We investigated components of the lectin pathway of complement activation in the context of sepsis at both genetic and protein levels in neonates, infants and older children. Major components of the lectin pathway and two genes for Toll-like receptors were studied in 87 neonates with confirmed sepsis and compared with 40 babies with infections who did not develop sepsis (disease controls) and 273 infection-free neonatal controls. A second cohort comprised 47 older children with sepsis and 87 controls. Low MBL-conferring genotypes (LXA/O+O/O) were more frequent in sepsis patients than in healthy controls but no significant differences in the frequency of SNPs of other lectin pathway genes (FCN1, FCN2, FCN3, MASP1/3, MASP2) or TLR receptor genes (TLR2, TLR4) were found. One case of primary MASP-2 deficiency was found among healthy pre-terms and one neonate suffering from SIRS was heterozygous for the rare FCN1 gene mutation, +6658 G>A. Generally, sepsis was associated with low serum MBL and low ficolin-2 concentrations on admission. Among neonates, ficolin-1 and MASP-2 levels were elevated in sepsis relative to healthy, but not disease, controls. Unlike neonates, ficolin-3 and MASP-2 levels were lower in older patients than in healthy controls while no difference was found for ficolin-1. With the possible exception of MBL, inherited lectin pathway insufficiencies do not seem to predispose to sepsis, rather changes in protein concentrations reflect alterations in disease course.

Animal lectins: a historical introduction and overview.

Some proteins we now regard as animal lectins were discovered before plant lectins, though many were not recognised as carbohydrate-binding proteins for many years after first being reported. As recently as 1988, most animal lectins were thought to belong to one of two primary structural families, the C-type and S-type (presently known as galectins) lectins. However, it is now clear that animal lectin activity is found in association with an astonishing diversity of primary structures. At least 12 structural families are known to exist, while many other lectins have structures apparently unique amongst carbohydrate-binding proteins, although some of those "orphans" belong to recognised protein families that are otherwise not associated with sugar recognition. Furthermore, many animal lectins also bind structures other than carbohydrates via protein-protein, protein-lipid or protein-nucleic acid interactions. While animal lectins undoubtedly fulfil a variety of functions, many could be considered in general terms to be recognition molecules within the immune system. More specifically, lectins have been implicated in direct first-line defence against pathogens, cell trafficking, immune regulation and prevention of autoimmunity.

Mannan-binding lectin: clinical significance and applications.

Mannan-binding lectin (MBL) is a collectin (protein with both collagen-like and C-type lectin domains) synthesised in the liver and secreted into the bloodstream. Its plasma concentration is for the most part genetically determined by a series of allelic dimorphisms located both in the structural gene and in the promoter region. Genotypes made up of combinations of seven haplotypes are mainly responsible for a 1000-fold concentration variation found in human beings. MBL is a pattern recognition molecule able to bind repeating sugar arrays on many microbial surfaces, and can activate complement via associated serine proteases. A poorly defined proportion (roughly 10%) of the population with the lowest MBL concentrations is thought to be MBL insufficient and more vulnerable to a variety of infectious and noninfectious disorders. The evidence that MBL makes an important contribution to innate immunity, by increasing susceptibility to disease and/or affecting the course of disease, is discussed in detail. Preliminary results from MBL replacement therapy are encouraging, and extension of this approach to large-scale randomised clinical trials would provide solid evidence concerning the physiological significance of this protein.

No Strong Relationship Between Components of the Lectin Pathway of Complement and Susceptibility to Pulmonary Tuberculosis.

Mycobacterium tuberculosis (TB) may utilise the lectin complement pathway to facilitate entry into its niche within macrophages. Previous studies examining mannose-binding lectin (MBL) in patients with TB have been limited by failure to correlate genotype/phenotype relationships. This study investigated serum levels and genotypes of MBL, Ficolin-2, Ficolin-3 and MASP-2 in 168 patients with pulmonary tuberculosis and 168 age/sex-matched controls. Low serum levels of MBL and Ficolin-2 were defined using cut-offs previously identified in the literature. Median MBL serum levels were higher in TB patients than controls-1400 ng/ml (IQR 435-2520) vs 1030 ng/ml (350-2050), p = 0.02-but this was not mirrored by a difference in MBL haplotype frequencies (MBL deficient haplotypes were observed in 11.9 % of TB patients and 11.3 % of controls). Severe Ficolin-2 deficiency (<1200 ng/ml) was more frequent in TB than controls (7.1 vs 3.0 %, odds ratio 2.51 95 % CI 0.86-7.28, p = 0.1) but the difference was not statistically significant. No relationship between Ficolin-2, Ficolin-3 or MASP-2 genotypes or serum levels and TB were observed. No strong relationship between the lectin complement pathway and pulmonary tuberculosis was observed. Previous data linking high MBL serum levels with TB were likely due to an acute phase response rather than a true effect on disease susceptibility.

Serum ficolin-2 in hospitalised patients with community-acquired pneumonia.

Mannose binding lectin (MBL) and ficolins contribute to host defence through activation of the lectin pathway of complement. In this study, serum levels of ficolin-2 and MBL were determined in 276 patients with community-acquired pneumonia (CAP). MBL deficiency and ficolin-2 insufficiency were defined using previously validated cut-offs. No differences were observed in MBL or ficolin-2 between patients and controls. MBL-deficient patients (<500 ng/ml) were not at higher risk of 30-day mortality odds ratio (OR) 0.97 (0.38-2.48,p=0.9) or a composite outcome of mortality, mechanical ventilation, vasopressor support (MV/VS) or complications OR 0.89 (0.44-1.77, p=0.9). Although no significant relationship between ficolin-2 insufficiency and outcome was observed, very low ficolin-2 levels (<1,200 ng/ml) were associated with an OR 1.23 (0.15-10.1), p=0.6 for 30-day mortality, 3.05 (0.61-15.2, p=0.2) for MV/VS and OR 2.05 (0.52-8.1, p=0.2) for the composite outcome. Low serum levels of MBL and ficolin-2 are not associated with CAP susceptibility. The high frequency of ficolin-2 insufficiency in patients with severe CAP would justify a larger investigation of ficolin-2 as a modifier of CAP severity.

Birds, babies and blood.

This is an autobiographical review describing the author's career in immunology research and summarizing his current understanding of the areas involved. Contributions to autoimmunity, immune deficiency, transfusion immunology, HLA-disease associations, reproductive immunology, cellular therapies, and innate immunity are included; also discussion of medical research ethics and various research-related activities.

Mannan-binding lectin in malignancy.

Complement may play a dual role in cancer: it may contribute either to the development or to the inhibition of tumour growth. Its components may be candidate biomarkers facilitating the disease detection, its progress or effectiveness of therapy. Additionally, complement deficiencies may increase the risk of infections and contribute to the higher mortality, especially in patients undergoing aggressive chemotherapy. In this paper, possible cancer associations of one of the factors activating complement via the lectin pathway, mannan-binding lectin (MBL), are discussed.

The relationship between FCN2 genotypes and serum ficolin-2 (L-ficolin) protein concentrations from a large cohort of neonates.

The human FCN2 gene codes for ficolin-2 (L-ficolin), a major pattern recognition molecule and activator of the lectin pathway of complement. Seven single nucleotide polymorphisms of this gene were investigated in a large series of cord blood DNA samples. Mutations from the majority to the minority alleles at -602, -4 and +6359 were associated with an increase, while mutations at -986, -557, -64 and +6424 were associated with a decrease, in protein concentration. Full (7 loci) genotypes were obtained for 1229 unrelated neonates, 12 sets of twin siblings and one set of triplets. Forty-four separate genotypes were detected. Four genotypes accounted for more than half the unrelated neonates, and >90% had one of the 12 commonest genotypes. Genotypes were associated with significant differences in mean serum ficolin-2, but the intra-genotype concentration ranges were large and greater than the inter-genotype differences. Consequently, there were no associations between genotypes and low birthweight babies or perinatal infections, and only a weak relationship with preterm deliveries, despite all three adverse pregnancy features being significantly associated with serum ficolin-2 protein. FCN2 genotyping may be of value in clinical studies, but not as a substitute for total serum ficolin-2 protein measurement.

Mannose-binding lectin deficiency and disease severity in non-cystic fibrosis bronchiectasis: a prospective study.

BACKGROUND: Mannose-binding lectin (MBL) is a key component of innate immunity. MBL deficiency is common (10-30% of the general population depending on the definition used) and has been associated with disease progression in cystic fibrosis. We aimed to assess the effect of MBL deficiency on disease severity in non-cystic fibrosis bronchiectasis. METHODS: We recruited patients with non-cystic fibrosis bronchiectasis and age-matched and sex-matched controls at a specialist bronchiectasis clinic in Edinburgh, UK. We assessed MBL function with genotyping (low-expressing genotype [deficiency] defined as homozygosity for exon 1 mutations [YO/YO] or compound heterozygosity [XA/YO]; YA/YO and XA/XA genotypes were defined as intermediate-expressing with all other genotypes defined as high-expressing) and serum measurements (deficiency defined with two parameters: <500 ng/mL or <200 ng/mL). We assessed rates of exacerbation, chronic bacterial colonisation, and lung function during 4 years of follow-up. FINDINGS: We included 470 patients with bronchiectasis and 414 controls. MBL genotype frequencies and MBL serum concentrations did not differ between patients and controls. 55 (12%) patients with bronchiectasis had low-expressing genotypes. These patients had a mean of 2·7 exacerbations per year (SD 1·8), compared with 1·9 per year (1·2) for 135 patients with intermediate-expressing genotypes and 1·9 per year (1·3) for 280 patients with high-expressing genotypes (p<0·0001). Chronic colonisation with bacteria was most frequent in patients with low-expressing genotypes (47 [85%] patients vs 82 [61%] patients with intermediate-expressing genotypes and 183 [65%] patients with high-expressing genotypes; p=0·0041); especially P aeruginosa colonisation (19 [35%] patients vs 13 [10%] patients and 36 [13%] patients; p<0·0001). Patients with low-expressing genotypes were more likely to be admitted to hospital for severe exacerbations during follow-up (27 [49%] patients vs 42 [31%] patients and 87 [31%] patients; p=0·032). Patients with low-expressing genotypes also had increased scores for radiological severity and worse quality of life compared with the other two groups. MBL serum deficiency (<200 ng/mL) was associated with increased exacerbations, hospital admissions, and radiological severity. When <500 ng/mL was used as the definition of deficiency, the associations with exacerbation frequency and radiological severity were no longer significant. INTERPRETATION: MBL might be an important modifier of disease severity in non-CF bronchiectasis. FUNDING: UK Medical Research Council, UK Chief Scientists Office.

H-ficolin (ficolin-3) concentrations and FCN3 gene polymorphism in neonates.

Serum H-ficolin (ficolin-3) concentrations (n=613) and FCN3 genotypes (n=529) from a large group of neonates are presented. Both pre-term deliveries and low birthweight (independently of gestational age) were significantly associated with low H-ficolin concentrations but not with heterozygosity for the FCN3 1637delC frameshift mutation. The presence of the variant allele, however, apparently influenced the protein level. No association of FCN3 gene heterozygosity or relative functional H-ficolin insufficiency (determined as serum level ≤8.6 µg/ml) with perinatal infections was found. One premature newborn, with confirmed infection caused by Streptococcus agalactiae, was H-ficolin-deficient (FCN3 variant homozygote, no detectable protein). We present what is only the fourth case report of total H-ficolin deficiency in the world literature. This neonate was however previously found to be mannan-binding lectin (MBL) as well as MBL-associated serine protease-2 (MASP-2) deficient and also had low serum L-ficolin.