Low Levels Of Mannan-binding Lectin Research Articles

Association of complement pathways with COVID-19 severity and outcomes

ObjectivesComplement activation has been implicated in COVID-19 pathogenesis. This study aimed to assess the levels of complement activation products and full-length proteins in hospitalized patients with COVID-19, and evaluated whether complement pathway markers are associated with outcomes. MethodsLongitudinal measurements of complement biomarkers from 89 hospitalized adult patients, grouped by baseline disease severity, enrolled in an adaptive, phase 2/3, randomized, double-blind, placebo-controlled trial and treated with intravenous sarilumab (200 mg or 400 mg) or placebo (NCT04315298), were performed. These measurements were then correlated with clinical and laboratory parameters. ResultsAll complement pathways were activated in hospitalized patients with COVID-19. Alternative pathway activation was predominant earlier in the disease course. Complement biomarkers correlated with multiple variables of multi-organ dysfunction and inflammatory injury. High plasma sC5b-9, C3a, factor Bb levels, and low mannan-binding lectin levels were associated with increased mortality. Sarilumab treatment showed a modest inhibitory effect on complement activation. Moreover, sera from patients spontaneously deposited C5b-9 complex on the endothelial surface ex vivo, suggesting a microvascular thrombotic potential. ConclusionThese results advance our understanding of COVID-19 disease pathophysiology and demonstrate the importance of specific complement pathway components as prognostic biomarkers in COVID-19.

Low circulating mannan-binding lectin levels correlate with increased frequency and severity of febrile episodes in myeloma patients who undergo ASCT and do not receive antibiotic prophylaxis.

Patients with multiple myeloma (MM) who undergo autologous stem cell transplantation (ASCT) are susceptible to severe infections. Low levels of circulating mannan-binding lectin (MBL) are associated with increased risk of infection. In this prospective study, we evaluated 100 patients who underwent ASCT regarding the effect of MBL on the incidence and severity of febrile episodes. Seventeen patients had MBL levels <500 ng/mL (11 received antibiotic prophylaxis and 6 did not). Although there was no statistical difference regarding the development of febrile episodes between patients with low and normal MBL, among 17 patients with low MBL levels, six out of eleven patients who received antibiotic prophylaxis developed a febrile episode compared with six out of six patients who did not receive antibiotic prophylaxis and developed a febrile episode. Patients with low MBL levels who responded less frequently to first line antibiotic therapy required more frequent administration of a second more advanced line of antibiotics, independently of receiving or not prophylaxis, and required prolonged hospitalization. In the univariate analysis low MBL associated with shorter OS. Our results suggest that patient with low MBL levels should receive antibiotic prophylaxis to reduce the number of febrile episodes and raise the issue of MBL replacement for these patients.

Genetically Determined MBL Deficiency Is Associated with Protection against Chronic Cardiomyopathy in Chagas Disease.

Chagas disease (CD) is caused by Trypanosoma cruzi, whose sugar moieties are recognized by mannan binding lectin (MBL), a soluble pattern-recognition molecule that activates the lectin pathway of complement. MBL levels and protein activity are affected by polymorphisms in the MBL2 gene. We sequenced the MBL2 promoter and exon 1 in 196 chronic CD patients and 202 controls. The MBL2*C allele, which causes MBL deficiency, was associated with protection against CD (P = 0.007, OR = 0.32). Compared with controls, genotypes with this allele were completely absent in patients with the cardiac form of the disease (P = 0.003). Furthermore, cardiac patients with genotypes causing MBL deficiency presented less heart damage (P = 0.003, OR = 0.23), compared with cardiac patients having the XA haplotype causing low MBL levels, but fully capable of activating complement (P = 0.005, OR = 7.07). Among the patients, those with alleles causing MBL deficiency presented lower levels of cytokines and chemokines possibly implicated in symptom development (IL9, p = 0.013; PDGFB, p = 0.036 and RANTES, p = 0.031). These findings suggest a protective effect of genetically determined MBL deficiency against the development and progression of chronic CD cardiomyopathy.

L-Asparaginase lowers plasma antithrombin and mannan-binding-lectin levels: Impact on thrombotic and infectious events in children with acute lymphoblastic leukemia.

L-asparaginase, a key therapeutic agent in the management of patients with acute lymphoblastic leukemia (ALL), dramatically impairs hepatic protein synthesis. We investigated the effects of prolonged exposure to L-asparaginase on antithrombin (AT), fibrinogen and mannan-binding-lectin (MBL) levels, and on the occurrence of thrombotic events (TE) and febrile neutropenia episodes (FN) in pediatric patients. Protein levels were measured in 97 children during 30 weeks of chemotherapy with L-asparaginase and up to 1 year following remission. TE and FN episodes were recorded during this period. Median AT level decreased from 0.96 IU/mL prior to treatment (range: 0.69-1.38) to 0.55 IU/mL (0.37-0.76) during therapy. Fibrinogen and MBL decreased from 3.18 g/L (1.29-7.28) and 1,177 ng/mL (57-5,343) to 1.56 g/L (0.84-2.13) and 193 ng/mL (57-544), respectively. All three proteins had recovered 1-4 weeks after L-asparaginase cessation. TE were reported in 22 (23%) patients. Of these, 11 occurred after a median of 10 administrations of L-asparaginase. Fifty-one FN were associated with infections, of which 36 occurred during treatment with L-asparaginase. Patients with low levels of MBL at diagnosis were at higher risk of FN associated with infections (RR = 1.59, 95%CI: 1.026-2.474). Both AT and MBL decreases were moderately correlated with fibrinogen (r = 0.51 and 0.58, respectively). Children with ALL are exposed to significant decrease in AT, fibrinogen and MBL levels, and concomitant increased risk of thrombosis and FN with infection during L-asparaginase treatment. Measuring plasma levels of these liver-derived proteins could help predict the occurrence of adverse events.

Mannan-binding lectin polymorphisms and serum levels in patients with endometriosis

ObjectiveTo investigate a possible association between endometriosis and low levels of mannan-binding lectin (MBL). Study designCase-control study of blood samples from 100 patients with endometriosis compared with results from a group of 350 blood donors. ResultThe frequency of MBL levels <100ng/ml was 14.0% in patients and 14.9% in controls. Correspondingly, the frequencies of low producing MBL genotypes were 15.0% and 16.0%, respectively. ConclusionNo association was found between endometriosis and low levels of MBL.

Association between Low Levels of Mannan-Binding Lectin and Markers of Autoimmune Thyroid Disease in Pregnancy

Functional deficiency of mannan-binding lectin (MBL) has been associated with adverse pregnancy outcome. Adverse events during pregnancy have also been described in women with autoimmune thyroid diseases (AITD), and thyroid hormones have been shown to influence serum levels of MBL. Therefore, the aim of this study was to analyse the impact of MBL-deficiency on the outcome of pregnancy in relation to the presence of AITD. Almost one year after delivery, we assessed serum MBL levels and MBL2-genotypes in 212 women positively screened for AITD in pregnancy. In 103 of these women, we could also measure MBL levels in frozen serum samples from the 9-12th gestational week, obtaining 96 pairs of MBL values (pregnancy vs. follow-up). As controls, 80 sera of pregnant women screened negatively for AITD were used. MBL2-genotyping was performed using multiplex PCR. Women with thyroid dysfunction and/or thyroid peroxidase antibodies (TPOAb) had lower MBL levels during pregnancy than controls, (3275 vs. 5000 ng/ml, p<0.05). The lowest levels were found in women with elevated thyroid-stimulating hormone (TSH) levels in the absence of TPOAb (2207 ng/ml; p<0.01 as compared to controls). MBL2 genotype distribution did not differ between subgroups. At a median follow-up period of 17 months (range: 3–78 months) after delivery, median MBL level had decreased further to 1923 ng/ml (p<0.0001) without significant changes in TSH. In an explorative survey, functional MBL-deficiency was neither linked to a history of spontaneous abortion, nor other obstetric complications, severe infections throughout life/pregnancy or antibiotics use in pregnancy. In conclusion, hypothyroidism during pregnancy is associated with decreased MBL levels, and the levels decreased further after delivery.

Low Levels of Mannan-Binding Lectin or Ficolins Are Not Associated with an Increased Risk of Cytomegalovirus Disease in HIV-Infected Patients

BackgroundIn HIV-infected patients, prediction of Cytomegalovirus (CMV) disease remains difficult. A protective role of mannan-binding lectin (MBL) and ficolins against CMV disease has been reported after transplantation, but the impact in HIV-infected patients is unclear.MethodsIn a case-control study nested within the Swiss HIV Cohort Study, we investigated associations between plasma levels of MBL/ficolins and CMV disease. We compared HIV-infected patients with CMV disease (cases) to CMV-seropositive patients without CMV disease (controls) matched for CD4 T-cells, sampling time, and use of combination antiretroviral therapy. MBL and M-ficolin, L-ficolin, and H-ficolin were quantified using ELISA.ResultsWe analysed 105 cases and 105 matched controls. CMV disease was neither associated with MBL (odds ratio [OR] 1.03 per log10 ng/mL increase (95% CI 0.73–1.45)) nor with ficolins (OR per log10 ng/mL increase 0.66 (95% CI 0.28–1.52), 2.34 (95% CI 0.44–12.36), and 0.89 (95% CI 0.26–3.03) for M-ficolin, L-ficolin, and H-ficolin, respectively). We found no evidence of a greater association between MBL and CMV disease in patients with low CD4 counts; however in the multivariable analysis, CMV disease was more likely in patients with an increased HIV RNA (OR 1.53 per log10 copies/mL; 95% CI 1.08–2.16), or a shorter duration of HIV-infection (OR 0.91 per year; 95% CI 0.84–0.98).ConclusionsCMV disease is not associated with low levels of MBL/ficolins, suggesting a lack of a protective role in HIV-infected patients.

Low levels of mannan-binding lectin or ficolins are not associated with an increased risk of cytomegalovirus disease in HIV-infected patients – A case–control study nested within the Swiss HIV Cohort Study

Low levels of mannan-binding lectin or ficolins are not associated with an increased risk of cytomegalovirus disease in HIV-infected patients – A case–control study nested within the Swiss HIV Cohort Study

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.

Mannan‐Binding Lectin (MBL) and MBL‐Associated Serine Protease 2 (MASP‐2) Genotypes in Colorectal Cancer

Mannan-binding lectin (MBL) and MBL-associated serine protease 2 (MASP-2) are key factors of the lectin pathway of complement activation. Polymorphisms of the MBL2 and MASP-2 genes affect serum levels of MBL and MASP-2. In patients with colorectal cancer (CRC), the MBL and MASP-2 serum levels are increased and high MASP-2 levels are associated with recurrence and poor survival, whereas low MBL levels predict post-operative pneumonia. It is not known whether these associations are genetically based. In this study, the MBL and MASP-2 genotypes are investigated in 593 patients with CRC and 348 healthy controls. The potential association between genetic profile and infections, recurrence and survival is evaluated. Four single-nucleotide polymorphisms (SNPs) of MBL2 were analysed using TaqMan assays, with characterization of MBL2 wildtype A, variants B, C and D and alleles H/L, Y/X and P/Q. The SNP D120G for MASP-2 was determined. Serum levels of MBL and MASP-2 were measured. The MBL2 and MASP-2 genotype distribution was similar among patients with CRC and healthy controls and MBL2 genotype significantly associated with MBL concentration in serum (P<0.0001). No significant association between MBL2/MASP-2 genotype and post-operative infectious complications (P=0.33 and 0.22), recurrent cancer or survival (P=0.74 and P=0.61 respectively) was found. Thus, the increased serum levels of MBL and MASP-2 found in patients with CRC are not explained for by genetic profiles. In contrast to what has been demonstrated for serum levels of MBL and MASP-2, the genotypes do not predict disease course of the CRC patients.

Infections of People with Complement Deficiencies and Patients Who Have Undergone Splenectomy

The complement system comprises several fluid-phase and membrane-associated proteins. Under physiological conditions, activation of the fluid-phase components of complement is maintained under tight control and complement activation occurs primarily on surfaces recognized as "nonself" in an attempt to minimize damage to bystander host cells. Membrane complement components act to limit complement activation on host cells or to facilitate uptake of antigens or microbes "tagged" with complement fragments. While this review focuses on the role of complement in infectious diseases, work over the past couple of decades has defined several important functions of complement distinct from that of combating infections. Activation of complement in the fluid phase can occur through the classical, lectin, or alternative pathway. Deficiencies of components of the classical pathway lead to the development of autoimmune disorders and predispose individuals to recurrent respiratory infections and infections caused by encapsulated organisms, including Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae. While no individual with complete mannan-binding lectin (MBL) deficiency has been identified, low MBL levels have been linked to predisposition to, or severity of, several diseases. It appears that MBL may play an important role in children, who have a relatively immature adaptive immune response. C3 is the point at which all complement pathways converge, and complete deficiency of C3 invariably leads to severe infections, including those caused by meningococci and pneumococci. Deficiencies of the alternative and terminal complement pathways result in an almost exclusive predisposition to invasive meningococcal disease. The spleen plays an important role in antigen processing and the production of antibodies. Splenic macrophages are critical in clearing opsonized encapsulated bacteria (such as pneumococci, meningococci, and Escherichia coli) and intraerythrocytic parasites such as those causing malaria and babesiosis, which explains the fulminant nature of these infections in persons with anatomic or functional asplenia. Paramount to the management of patients with complement deficiencies and asplenia is educating patients about their predisposition to infection and the importance of preventive immunizations and seeking prompt medical attention.

Influence of mannose-binding lectin genotypes and serostatus in allo-SCT: analysis of 131 recipients and donors

Mannan-binding lectin (MBL) deficiency is determined by MBL gene polymorphisms and is associated with an increased infection risk. To clarify the role of MBL in Allo-SCT, 131 recipients-donors were analysed. MBL genotypes were determined by PCR and heteroduplex analyses, MBL serum levels by ELISA, and MBL oligomers by western blotting. MBL levels <400 ng/ml were associated with increased susceptibility to fungal pneumonia (7/12 vs 35/111; P=0.04, adjusted P=0.002), HSV/VZV (7/12 vs 26/111; P=0.03), CMV reactivation and acute GVHD. Donor genotypes had no influence. Pre-SCT MBL levels corresponded to recipients' genotypes (P<0.001), changed significantly post-SCT, but were not influenced by donors' genotypes. MBL oligomer profiles were similar pre-/post-SCT. Cultured CD34+ cells were found not to synthesise MBL. In conclusion, low MBL levels pre-transplant predisposed patients to sepsis, fungal and viral infection. Donors' MBL genotypes did not influence infection rates. Prospective studies should clarify the importance of MBL as a prelude for MBL replacement after SCT.

Association of three systemic lupus erythematosus susceptibility factors, PD‐1.3A, C4AQ0, and low levels of mannan‐binding lectin, with autoimmune manifestations in icelandic multicase systemic lupus erythematosus families

To study autoimmune diseases and autoantibodies in Icelandic multicase systemic lupus erythematosus (SLE) families and to determine the association of 3 SLE susceptibility factors, PD-1.3A, C4AQ0, and low levels of mannan-binding lectin (MBL), with autoimmune disease in this population. Eight SLE multicase families were studied, comprising a total of 124 family members (23 patients with SLE and 101 relatives). The diagnosis of an autoimmune disease was established and autoantibodies were measured in each family. In addition, PD-1.3A alleles were genotyped, and C4AQ0 allotypes were established by electrophoresis and haplotype analysis. Low levels of MBL were determined using enzyme-linked immunosorbent assay and variant-allele genotyping. In the SLE multicase families there was a high frequency of other autoimmune diseases (32.2%) and a high frequency of autoantibodies (53.2%). Of all family members, 59.7% were determined to have SLE, other autoimmune diseases, antinuclear antibodies, and/or other autoantibodies. The families showed genetic heterogeneity for PD-1.3A, C4AQ0, and low MBL levels; the frequency of each factor ranged from 0% to 85%. The frequencies of PD-1.3A and C4AQ0 were significantly increased in patients with SLE, relatives with other autoimmune diseases, and non-autoimmune disease relatives compared with controls. In the 7 families whose members had low levels of MBL, this factor was significantly associated with SLE, but the frequency of low MBL was decreased in relatives with other autoimmune diseases as compared with non-autoimmune disease relatives and controls. There were indications of an additive effect, and 91% of patients with SLE, 78% of relatives with other autoimmune diseases, and 75% of non-autoimmune disease relatives carried at least 1 of the 3 factors. These results demonstrate a high frequency of autoimmune diseases and autoantibodies in SLE multicase families. PD-1.3A and C4AQ0 are part of a predisposing genetic background. Other genetic and/or environmental factors are necessary for disease expression, demonstrated by a high frequency of PD-1.3A and C4AQ0 in non-autoimmune disease relatives. Low MBL levels may be one such contributing factor. The results of this study provide an example of epistatic genetic effects and overlapping genetics in autoimmune diseases.

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 1124ng/ml, respectively, while median LP activity was 272mU/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.

Insulin Resistance Is Associated With Decreased Circulating Mannan-Binding Lectin Concentrations in Women With Polycystic Ovary Syndrome

Insulin resistance has been described as associated with low levels of mannan-binding lectin (MBL), a protein of the innate immune system (1). The aim of the present study was to study serum MBL concentration in polycystic ovary syndrome (PCOS) in relation to insulin resistance, adiponectin, and high-sensitivity C-reactive protein (hsCRP). Insulin sensitivity, serum MBL concentration, adiponectin, hsCRP, and sex hormones were measured in 102 PCOS (34 of whom were lean and 68 overweight or obese) …

Mannan-binding lectinMBL2gene polymorphism in chronic hepatitis C: association with the severity of liver fibrosis and response to interferon therapy

Hepatitis C virus (HCV) is a major cause of hepatic disease and of liver transplantation worldwide. Mannan-binding lectin (MBL), encoded by the MBL2 gene, can have an important role as an opsonin and complement activating molecule in HCV persistence and liver injury. We assessed the MBL2 polymorphism in 102 Euro-Brazilian patients with moderate and severe chronic hepatitis C, paired for gender and age with 102 HCV seronegative healthy individuals. Six common single nucleotide polymorphisms in the MBL2 gene, three in the promoter (H/L, X/Y and P/Q) and three in exon 1 (A, the wild-type, and B, C or D also known as O) were evaluated using real-time polymerase chain reaction with fluorescent hybridization probes. The concentration of MBL in plasma was measured by enzyme-linked immunosorbent assay. The frequency of the YA/YO genotype was significantly higher in the HCV patients compared with the controls (P = 0.022). On the other hand, the genotypes associated with low levels of MBL (XA/XA, XA/YO and YO/YO) were decreased significantly in the patients with severe fibrosis (stage F4), when compared with the patients with moderate fibrosis (stage F2) (P = 0.04) and to the control group (P = 0.011). Furthermore, MBL2 genotypes containing X or O mutations were found to be associated with non-responsiveness to pginterferon and ribavirin treatment (P = 0.023). MBL2 polymorphisms may therefore be associated not only with the development of chronic hepatitis C, but also with its clinical evolution and response to treatment.

Association of deficiency for mannan-binding lectin with anti-mannan antibodies in Crohnʼs disease: A family study

Antibodies against mannan, a component of the yeast Saccharomyces cerevisiae cell wall, are more frequently found in Crohn's disease (CD) patients with low levels of mannan-binding lectin (MBL). MBL concentration depends on genetic polymorphisms. The aim of this study was to evaluate whether low MBL is related to ASCA production in healthy family members of CD patients. ASCA and MBL concentrations in sera from patients (n=52), and their 158 healthy relatives were measured by enzyme-linked immunosorbent assay (ELISA). Genetic MBL variants were determined by DNA sequencing. Thirty-five (67%) patients were ASCA-positive. Twenty-six (74%) of the 35 ASCA-positive patients had low MBL levels (<500 ng/mL), whereas only 4 (24%) of the 17 ASCA-negative patients had low values for MBL (P=0.001). ASCA were found in 38 (24%) family members. Twenty-three (50%) of 46 family members with low values for MBL were ASCA-positive compared to 15 (13%) of 112 family members with normal values for MBL (P<0.0001). ASCA were found in 33 of 104 (32%) family members of ASCA-positive patients and in 5 family members (9%) of ASCA-negative patients (P=0.002). Relatives with mutations leading to MBL deficiency had significantly more frequent ASCA than relatives without these mutations (P=0.018). MBL deficiency is associated with ASCA positivity not only in patients with CD, but also in their relatives. An impaired innate immune system defined by low MBL serum concentrations may lead to an increased reactivity of the specific immune system to mannan antigens, and therefore facilitate the generation of ASCA.

Second‐generation nanofiltered plasma‐derived mannan‐binding lectin product: process and characteristics

Mannan-binding lectin (MBL) is an important component of the innate immune defence; it binds to carbohydrate structures on pathogenic micro-organisms resulting in complement activation and opsonization. Individuals with low MBL levels are at risk of recurrent and severe infections. Substitution therapy with plasma-derived MBL is a promising treatment of diseases associated with MBL deficiency. A first-generation MBL product has been shown to be safe and well tolerated, and patients have benefited from MBL treatment. Following is a description of the development of a nanofiltered second-generation MBL product from Cohn fraction III, with the use of a new affinity matrix for MBL purification and the characteristics of this improved product. Carbohydrate-based gels were comparatively screened as affinity matrices. MBL was extracted from fraction III, and affinity purified on a Superdex 200 pg column. The eluted material underwent two virus reduction steps: filtration through Planova 20N and solvent/detergent treatment. It was further purified by anion-exchange and gel-filtration chromatography. The affinity eluate and the final MBL fraction were characterized by protein chemical, immunological, and functional assays. In production scale, Superdex 200 pg was found to be superior to other carbohydrate-based matrices, and MBL was affinity purified from fraction III with a yield of 70%. The viral safety was increased by performing a nanofiltration of the affinity eluate through Planova 20N with a minimal loss of MBL. The purity of the final MBL fraction was 53% excluding the MBL-associated serine proteases (MASP). The product consisted of high-oligomeric MBL, with two dominating forms, and with MASP-1, -2, -3 and 19 kDa MBL-associated protein (MAp19). Only a few protein impurities were present, the major being alpha2-macroglobulin. MBL formed complexes with alpha2-macroglobulin bridged by MASP-1 covalently attached to the latter. The functional activity, assessed by mannan-binding activity and opsonic function, was intact, whereas half of the C4 activating capacity was lost during the production process. A second-generation MBL process was developed with an average yield of 50%. It was possible to nanofilter the MBL-MASP complexes through Planova 20N with only a minor loss resulting in an increased safety profile of this MBL product.

Phase I Safety, Tolerability, and Pharmacokinetic Study of Recombinant Human Mannan-Binding Lectin

Mannan-binding lectin (MBL), a human plasma protein, plays an important role in the innate immune defence. MBL recognizes microorganisms through surface carbohydrate structures. Due to genetic polymorphisms, MBL plasma concentrations range from 5 to 10,000 ng/mL. Approximately 30% of the human population have low levels of MBL (below 500 ng/mL). MBL deficiency is associated with increased susceptibility to infections in immunosuppressed individuals, e.g., during chemotherapeutically induced neutropenia. Replacement therapy with MBL may be beneficial in this patient group, and recombinant human MBL (rhMBL) is in development as a novel therapeutic approach. To assess the safety, tolerability, and pharmacokinetics of rhMBL, a placebo-controlled double-blinded study was performed in MBL-deficient healthy male subjects. rhMBL was administered as both single intravenous (i.v.) infusions (0.01, 0.05, 0.1, and 0.5 mg/kg) and repeated i.v. infusions (0.1 or 0.3 mg/kg given at 3-day intervals). There were no difference in incidence and type of adverse events reported in the study between the groups of subjects receiving rhMBL and the placebo group. All adverse events reported as drug-related were mild and no serious adverse events were recorded. There were no clinically significant changes in laboratory evaluations, ECG or vital signs, and no anti-MBL antibodies were detected following rhMBL administration. After single i.v. doses of rhMBL the maximal plasma levels increased in a dose-dependent manner reaching a geometric mean of 9710 ng/mL+/-10.5% in the highest dose group (0.5 mg/kg), with an elimination half-life of approximately 30 h. No rhMBL accumulation in plasma was observed following repeat dosing. Administration of rhMBL restored the ability to activate the MBL pathway of the complement system without non-specific activation of the complement cascade. In conclusion, no safety or tolerability concern was raised following rhMBL administration no signs of immunogenicity detected, and an rhMBL plasma level judged sufficient to achieve therapeutic benefit (>1000 ng/mL) can be achieved.

Thyroid hormone increases mannan-binding lectin levels

Recent studies have indicated the existence of causal links between the endocrine and immune systems and cardiovascular disease. Mannan-binding lectin (MBL), a protein of the innate immune system, may constitute a connection between these fields. To test whether thyroid hormone regulates MBL levels, we studied eight patients with Graves' hyperthyroidism before and after methimazole therapy, eight healthy subjects before and after short-term experimental hyperthyroidism, and eight hypothyroid patients with chronic auto-immune thyroiditis before and after L-thyroxine substitution. In all hyperthyroid patients, MBL levels were increased--median (range), 1886 ng/ml (1478-7344) --before treatment and decreased to 954 ng/ml (312-3222) after treatment (P = 0.01, paired comparison: Wilcoxon's signed ranks test). The healthy subjects had MBL levels of 1081 ng/ml (312-1578). Administration of thyroid hormones to these persons induced mild hyperthyroidism and increased MBL levels significantly to 1714 ng/ml (356-2488) (P = 0.01). Two of the eight hypothyroid patients had undetectably low levels of MBL both before and after L-thyroxine substitution. The other six hypothyroid patients had decreased levels of MBL of 145 ng/ml (20-457) compared with 979 ng/ml (214-1533) after L-thyroxine substitution (P = 0.03, paired comparison: Wilcoxon's signed ranks test). Our data show that thyroid hormone increases levels of MBL. MBL is part of the inflammatory complement system, and this modulation of complement activation may play a role in the pathogenesis of a number of key components of thyroid diseases.