Jens Chr. Jensenius

Deficiency of Mannose-Binding Lectin Greatly Increases Susceptibility to Postburn Infection with Pseudomonas aeruginosa

Burn injury disrupts the mechanical and biological barrier that the skin presents against infection by symbionts like the Pseudomonas aeruginosa, a Gram-negative bacteria. A combination of local factors, antimicrobial peptides, and resident effector cells form the initial response to mechanical injury of the skin. This activity is followed by an inflammatory response that includes influx of phagocytes and serum factors, such as complement and mannose-binding lectin (MBL), which is a broad-spectrum pattern recognition molecule that plays a key role in innate immunity. A growing consensus from studies in humans and mice suggests that lack of MBL together with other comorbid factors predisposes the host to infection. In this study we examined whether MBL deficiency increases the risk of P. aeruginosa infection in a burned host. We found that both wild-type and MBL null mice were resistant to a 5% total body surface area burn alone or s.c. infection with P. aeruginosa alone. However, when mice were burned then inoculated s.c. with P. aeruginosa at the burn site, all MBL null mice died by 42 h from septicemia, whereas only one-third of wild-type mice succumbed (p = 0.0005). This result indicates that MBL plays a key role in containing and preventing a systemic spread of P. aeruginosa infection following burn injury and suggests that MBL deficiency in humans maybe a premorbid variable in the predisposition to infection in burn victims.

Recombinant expression of human mannan-binding lectin

Mannan-binding lectin (MBL) constitutes an important part of the innate immune defence by effecting the deposition of complement on microbial surfaces. MBL deficiency is among the most common primary immunodeficiencies and is associated with recurrent infections and symptoms of poor immune complex clearance. Plasma-derived MBL has been used in reconstitution therapy but concerns over viral contamination and production capacity point to recombinant MBL (rMBL) as a future source of this protein for clinical use. Natural human MBL is an oligomer of up to 18 identical polypeptide chains. The synthesis of rMBL has been accomplished in several mammalian cell lines, however, the recombinant protein differed structurally from natural MBL. In this, study we compare rMBL produced in myeloma cells, Chinese hamster ovary (CHO) cells, human hepatocytes, and human embryonic kidney (HEK) cells. We report that rMBL structurally and functionally similar to natural MBL can be obtained through synthesis in the human embryonic kidney cells followed by selective carbohydrate affinity chromatography.

New insights on the structural/functional properties of recombinant human mannan-binding lectin and its variants.

Inefficient activation of complement lectin pathway in individuals with variant mannan-binding lectin (MBL) genotypes has been attributed to poor formation of higher order oligomers by MBL. But recent studies have shown the presence of large oligomers of MBL (approximately 450 kDa) in serum of individuals with variant MBL alleles. The recombinant forms of MBL (rMBL) variants except MBL/B that assemble into higher order oligomers have not yet been reported. In the present study, structural/functional properties of recombinant forms of wild type MBL (rMBL/A) and its three structural variants, rMBL/B, C, and D generated in insect cells were examined. Western blot analysis indicated covalently linked monomers to hexamers while gel filtration chromatography exhibited non-covalently linked higher order oligomers in addition to prevalent low oligomeric forms. Mannan binding determined by ELISA showed rMBL/A but not the structural variants bind to mannan. Apparent avidity of monoclonal antibody used was found to be about 18- to 52-fold weaker for rMBL structural variants than rMBL/A. Complement activation varied with maximum impairment apparent in rMBL/C followed by rMBL/B, but rMBL/D was functional to the same extent as rMBL/A. Comparison of rMBL/A to MBL purified from plasma (pMBL/A) indicated 8- and 24-fold weaker binding to mannan by BIAcore analysis and ELISA and about 5-fold lesser efficiency in activating complement. The findings provide new insights on the structural/functional properties of rMBL variants and imply that lectin pathway activation may be impaired in individuals, homozygous for the mutant alleles, MBL/C and to a lesser extent MBL/B but not MBL/D.

A method for blocking antigen-independent binding of human IgM to frozen tissue sections when screening human hybridoma antibodies.

Using an indirect immunoperoxidase technique antigen independent binding of both human monoclonal and polyclonal IgM was found to a wide range of frozen sections of normal and malignant human glandular epithelia. Identical binding was found using dimeric human IgA (dIgA), whereas no binding was found with monomeric human IgA or human IgG. Secretory component (SC) was found to be the component in these tissues mediating antigen-independent binding of human IgM and dIgA antibodies. A method for the blocking of this antigen-independent binding of human IgM and dIgA was evaluated. Frozen sections of tissues containing SC were blocked with antibody to endogenous immunoglobulin and preincubated with rabbit anti-secretory component antibody (anti-SC) before applying the human monoclonal antibody. This treatment blocked the binding of control polyclonal and monoclonal human IgM to sections of SC-containing tissues such as respiratory and colonic epithelia. The influence of anti-SC on the binding of dIgA could not be established due to interactions between the anti-SC antibody and the IgA preparation. Using this method human hybridoma supernatants containing IgM could be readily screened for reactivity with frozen tissue sections from patients with colo-rectal cancer. This approach is recommended for the screening of human IgM monoclonal antibodies on frozen human tissue sections.

Human conglutinin-like protein

The presence in human plasma of a molecule homologous to bovine congtutinin is indicated by the results of biological and immunochemical analysis. The human conglutinin-like protein shows calcium-dependent binding to complement-treated solid phase IgG and immunological cross-reaction with chicken anti-bovine conglutinin. The binding of the human protein to complement-treated IgG was inhibited by N-acetyl-D-glucosamine but not by other sugars. Analysis by SDS-PAGE and Western blotting showed reaction of anticonglutinin with molecules of similar mobility to the monomer and hexamer of bovine conglutinin.

Difference and ratio plots: simple tools for improved presentation and interpretation of scientific data Unexpected possibilities for the use of the conglutinin binding assay in inflammatory rheumatic diseases

11111onglutinin-binding assay (KgBa) has gained widespread use for the detection of circulating immune complexes. A recent paper questioned the interpretation of the results obtained by this method and the validity of the assay (Holmskov et al. (1992) J. Immunol. Methods 148, 225). We now present hitherto unnoted differences between controls and patients with either rheumatoid arthritis or systemic lupus erythematosus. For this we use simple, but unconventional, graphic representations of the data, based on difference plots and ratio plots. Differences between patients with Burkitts lymphoma and systemic lupus erythematosus from another previously published study (Macanovic, M. and Lachmann, P.J. (1979) Clin. Exp. Immunol. 38, 274) are also represented using ratio plots. Our observations indicate that analysis by regression analysis may often be misleading.

Constant heavy-chain (CH1) and L-chain dependence of rabbit VH allotype determinants demonstrated by polyethylene glycol precipitation radioimmunoassays

Abstract A simple and reliable polyethylene glycol (PEG) precipitation radioimmunoassay is described and used for examining the expression of V H allotype determinants (al) on various immunoglobulins and fragments. The alloantisera were obtained by immunization with free or antigen-complexed IgG. The results demonstrate that most of the antibody reacts with determinants which are only fully expressed in the presence of the first constant domain of the H-chain (C H l γ ), or the J γ -segment, as well as the L-chain. Only a minor part of the antibody reacts with free V H or IgA. These results may have important implications for the use of alloantibody in studies of the V H -domain.

Chapter 9:Personal Accounts of the Discovery of MASP-2 and its Role in the MBL Pathway of Complement Activation

In a period spanning more than three decades, the MRC Immunochemistry Unit was to the authors of this chapter a ‘friendly giant’ in the sciences of the complement system that offered great support and guidance. Our account of how the mannan-binding lectin (MBL) associated serine proteases 2 and 3 (M...

Glycopeptide mimics of mammalian Man9GlcNAc2. Ligand binding to mannan-binding proteins (MBPs)

A novel and simple approach for rational design of oligosaccharide mimics has been developed. Mammalian high-mannose triantennary structure Man9GlcNac2 has been subjected to molecular modelling using the NMR data available on structural fragments of the oligosaccharide. The analysis indicated four different low energy conformations, and the spatial arrangement of terminal disaccharides of the oligosaccharide antennae were stimulated with glycopeptides carrying disaccharides by applying weak constraints between the saccharide parts in MD-simulations on a large array of tri- to octaglycopeptides. The five glycopeptides exhibiting the best fit with the four minimum energy confirmations of the oligosaccharide were synthesized by solid phase glycopeptide assembly using glycosylated fluoren-9-ylmethyloxycarbonyl-amino acid-O-pentafluorophenyl esters as building blocks. The glycan was acyl protected alpha-D-Man-(1-->2)-alpha-D-Man and Ser, Thr and Hyp were the glycosylated amino acids. The deprotected and purified glycopeptides were subjected to NMR analysis for characterization, and in order to investigate the cis-trans isomerism of the carbimide bonds to Hyp. The glycopeptides were tested for their ability to inhibit binding of mannan-binding protein to mannan from Saccharomyces cerevisiae. They were found to be weak inhibitors showing no indication of multivalent interaction with the mannan-binding protein.

Preparation of antigen-binding monomeric and half-monomeric fragments from human monoclonal IgM antibodies against colorectal cancer-associated antigens

The large size of human IgM monoclonal antibodies (MAbs) may impede the tumor-localizing capacity. A procedure is described for the preparation of antigen-binding monomeric (IgMm) and half-monomeric (IgM1/2m) fragments from two human IgM MAbs, COU-1 and D4213. The fragments retained binding activity against colon carcinoma. Six different reducing reagents (dithiothreitol, 2-mercaptoethanol, 2-mercaptoethylamine, L-cysteine, metabisulphite, ascorbic acid) were investigated over a range of concentrations, pHs, and incubation periods. The reduced IgM preparations were alkylated with iodoacetamide and fractionated by high-performance gel permeation chromatography. The fractions were directly collected on ELISA plates coated with extracts of colon cancer cells. Antigen-binding IgMm and IgM1/2m fragments were obtained after treatment with mercaptoethanol, mercaptoethylamine, metabisulphite, and cysteine. IgMm and IgM1/2m fragments were also obtained after dithiothreitol treatment. These fragments were, however, nonreactive. The pH during the reduction was important for optimal yields of the fragments. The fragments obtained with 2-mercaptoethanol and mercaptoethylamine were most effective in binding to the cancer cell extract. The association constants per binding site for intact, monomeric, and half-monomeric COU-1 were by competitive inhibition assays estimated at 1.5 x 10(8) M-1, 3.1 x 10(8) M-1 and 4.0 x 10(6) M-1, respectively. The reduction of human IgM MAbs to IgMm and IgM1/2m fragments may facilitate the tumor localization when these are used in the diagnosis and therapy of cancer patients.