David C. Kilpatrick

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.

Mannan-binding lectin insufficiency in children with recurrent infections of the respiratory system

Blood samples were collected over a 4‐year period from 335 children (aged 1–16 years) suffering from recurrent respiratory infections and 78 controls. The patients were subdivided into four groups: I, children with no immune system defects detected (n = 101); II, children with allergies (n = 94); III, children with humoral response defects (n = 93); and IV, children with disturbances of cellular immunity (n = 66). Nineteen patients had both humoral and cellular abnormalities. All patients and controls were investigated to determine the exon 1 and promoter region variants of the mbl‐2 gene. MBL serum concentrations were also determined in samples from 291 patients and 75 controls. The proportion of O (B, D or C) alleles was significantly higher in the patient group compared to controls, and this association was strongest for subgroup III. The promoter LX variant frequency was also commoner in the patients as a whole, and significantly so in subgroups II and IV. Genotypes markedly influenced MBL concentrations in all groups, and correlated with ability to activate the lectin pathway of complement activation. The strongest and most significant inverse correlations between serum MBL and respiratory disease were found in patient group III and in 17 patients with multiple humoral and/or cellular abnormalities. Among nine patients with unexpectedly low LP activity in view of their MBL concentrations, one person was found to be MASP‐2 deficient. Our results indicate that mannan‐binding lectin insufficiency, with or without a coexisting immune defect, is associated with the occurrence of recurrent respiratory infections in childhood, and this relationship is particularly strong and statistically significant in children with concomitant impairments of humoral immunity.

P35, an opsonic lectin of the ficolin family, in human blood from neonates, normal adults, and recurrent miscarriage patients

P35 (L-ficolin) is a novel calcium-dependent lectin which may have a role in innate immunity. The concentration range of P35 in blood donor sera was found to be 1.1-12.8 (median 3.7) microg/ml, with most values below 6.0 microg/ml. Sera from women with recurrent miscarriage were found to have a generally similar distribution of concentrations, but 3 (4%) had values lower than the lower limit of normal defined by the blood donor sera. Cord blood samples had significantly lower levels of P35 (median 2.5 microg/ml) than adults, and cord P35 concentrations correlated with gestational age. During ethanol fractionation of plasma, P35 was enriched in Cohn Fraction III, but did not bind to mannan immobilised on 3M-Emphaze. P35 thus has some similarities to, and some differences from, mannan binding lectin.

Establishment and characterization of a bank of cytotoxic T lymphocytes for immunotherapy of epstein-barr virus-associated diseases.

Adoptive immunotherapy using Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) generated ex vivo can be an effective treatment of EBV-positive posttransplantation lymphoproliferative disease (PTLD). We describe the establishment of a cryopreserved repository of allogeneic virus-specific CTL lines, to our knowledge the first of its kind in the world. CTL lines were grown by weekly stimulation with autologous EBV immortalized lymphoblastoid cell lines (LCLs) from 96 EBV-seropositive blood donors. Analysis of 60 CTL lines grown continuously for 7 to 10 weeks showed an average proportional weekly increase in cell numbers of 1.4, with an overall increase ranging from 1.1 to 83.4. The greatest increase occurred during the early culture period. After four rounds of stimulation, killing of autologous LCLs was generally high (mean 48%); however, most lines required 9 or 10 stimulations to reduce the killing of nonspecific targets. Overall, 79% of CTLs generated showed acceptable levels of specific killing. Phenotypically, the CTL lines consisted of TCR&agr;β+, CD8+ T cells (medians 97% and 90% respectively) with a minority population of CD4+ T cells (median 2%). Most cells expressed the activation and differentiation markers, HLA-DR, CD26, CD45RO, CD69, and CD150. Favorable results have been obtained in an open trial using partially HLA-matched, allogeneic CTLs from this bank to treat PTLD patients. This now represents a single resource that can provide therapeutic CTLs rapidly on a countrywide basis, superseding the time-consuming, expensive practice of generating autologous CTLs from each patient requiring treatment. Additionally, other patient groups, such as those with EBV-positive Hodgkin disease, may benefit from CTL treatment.

L‐ficolin in children with recurrent respiratory infections

The lectin pathway of complement activation is used by a collectin, mannan‐binding lectin (MBL), and two ficolins, L‐ficolin and H‐ficolin, to opsonize microorganisms for phagocytosis. We published evidence recently that MBL insufficiency is associated with recurrent respiratory infections in childhood. We have now measured serum L‐ficolin in 313 respiratory infection patients and 74 healthy control children. L‐ficolin concentrations below the lower limit of the control group were found in 6% of the patients (P < 0·02) and were associated most strongly with children having co‐existing atopic disorders (11%; P = 0·002). We suggest that L‐ficolin may have a role in protection from microorganisms complicating allergic disease.

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.

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-β-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.

L-ficolin (ficolin-2) insufficiency is associated with combined allergic and infectious respiratory disease in children

We previously reported an association between relative L-ficolin deficiency and recurrent respiratory infections co-existing with allergic disorders in children. To confirm and extend this preliminary finding, we performed a prospective study on children of a similar age (mean 8.9 years) designed to establish whether the principal relationship was with infection or allergy. Serum L-ficolin values in healthy children were normally distributed with a mean value of 3838 ng/ml. L-ficolin concentrations were generally lower in patients with asthma and/or allergic rhinitis with (mean 3413 ng/ml; p=0.02) or without (3512 ng/ml; p<0.07) respiratory infections, but not in patients with respiratory infections without allergic disease (3623 ng/ml; p=0.2). The lower average values in the group comprised of children with respiratory allergy and infections were largely due to a high proportion of very low values: 18.3% had values below 2150 ng/ml compared to only 5.5% of healthy controls (OR=3.9; p=0.01). This relationship was not apparent in the groups characterized by allergy without infection or infections without allergy. An association between mannan-binding lectin (MBL) insufficiency and recurrent respiratory infections was also confirmed. One of the patients was MASP-2 deficient, evidenced both by MASP2 genotyping and by lectin pathway activity measurement. In conclusion, L-ficolin may confer some protection from microorganisms that exacerbate allergic inflammation in the lung and its relative deficiency may contribute to enhanced susceptibility to respiratory infections. MBL insufficiency and MASP-2 deficiency are risk factors for recurrence of infections independently of allergic disease.

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.

Extremes of L-ficolin concentration in children with recurrent infections are associated with single nucleotide polymorphisms in the FCN2 gene.

l‐ficolin (also called ficolin‐2, P35 or hucolin) is a soluble pattern recognition molecule of suspected importance in anti‐microbial immunity. It activates the lectin pathway of complement and acts as an opsonin. l‐ficolin, encoded by the FCN2 gene, recognizes microbial polysaccharides and glycoconjugates rich in GlcNAc or GalNAc. We report here data concerning four single nucleotide polymorphisms (SNPs) of the FCN2 gene and their relationship to l‐ficolin serum concentrations. There are two pairs of SNPs in linkage disequilibrium: ss32469536 (located in promoter) with rs7851696 (in exon 8) and ss32469537 (promoter) with ss32469544 (exon 8). We selected groups possessing low or high serum l‐ficolin concentrations (≤ 2·8 µg/ml or ≥ 4·5 µg/ml, respectively) from Polish children suffering from recurrent respiratory infections (n = 146). Low l‐ficolin levels were associated with variant alleles for ss32469536 and rs7851696 and normal alleles for ss32469537 and ss32469544. Conversely, high l‐ficolin levels were associated with variant alleles of ss32469537 and ss32469544. FCN2 genotyping should be a valuable additional tool for disease association studies.