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The molecular basis of a common defect of opsonisation.

Super, Michael; (1990) The molecular basis of a common defect of opsonisation. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

A failure of serum to opsonise baker's yeast Saccharomyces cerevisiae is a defect found in 5-7% of the general population but at a higher frequency in paediatric patients with repeated, unexplained infections. Previous work had suggested an abnormality of the complement system and in the present studies assays were devised to measure the deposition of complement moieties on yeast zymosan and on the surface of microtitre plates coated with the yeast cell wall component - mannan. The latter was found to be technically superior and was used for all the subsequent studies. C4 fragments, properdin and Factor B were bound to the mannan-coated plates as well as the expected opsonic fragments C3b and C3bi. Analysis of 179 sera from healthy adult blood donors revealed that the binding levels of C3b, C4, properdin and Factor B were highly significantly correlated. When the assays were carried out at the same dilution in MgEGTA, there was no detectable binding of complement proteins to the mannan surface, confirming that no alternative pathway activation was occurring at this serum concentration in this experimental system. The levels of bound anti-mannan antibodies (IgG, IgA and IgM and the IgG subclasses IgG1, IgG2 and IgG3 ) were found to be completely unrelated to the C3bi levels previously observed. In parallel with these investigations affinity chromatography of serum with normal opsonisation on a mannan-Sepharose column suggested that a calcium dependent macromolecule with a molecular weight of 600-700 kDa was able to correct the opsonic defect. A candidate molecule having these physicochemical characteristics and known to be complement activating in the rat was the serum lectin mannose binding protein (MBP). Using a polyclonal monospecific rabbit anti-human MBP the protein was assayed in the blood donor population both by antibody capture ELISA procedure and the previously described mannan capture procedure. The levels of MBP bound in the latter correlated with the levels of C4, C3b, properdin and Factor B but not with the levels of anti-mannan immunoglobulins bound. The results suggest that, in this experimental system using low concentrations of serum, mannose binding protein initiates an antibody independent mechanism of cleavage of the classical pathway component C4 which subsequently regulates the degree of cleavage of C3 and binding of alternative pathway proteins. In a study of 10 paediatric patients previously shown to have the functional opsonic defect the median MBP concentration was 4.9μg/l (range 2.5-35μg/l) compared to 142μg/l for a paediatric control group (range 2.5-880μg/l). Purified MBP was shown to correct the opsonic deficiency in a dose dependent fashion in an in vitro assay measuring deposition of complement moieties on a mannan coated surface. Thus it appears that low levels of serum MBP underlie the common opsonic defect. Cross species functional correction assays were performed with sera from both eutherian mammals and marsupials. Partial or complete correction was observed in every case suggesting that MBP is a widely distributed conserved molecule with a major role in antigen non-specific immune responses to pathogens.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The molecular basis of a common defect of opsonisation.
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences
URI: https://discovery.ucl.ac.uk/id/eprint/10120608
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