Title:
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Autoantibodies in Multiple Sclerosis.
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rvIultiple sclerosis (MS) is an inflammatory disease of the central nervous system, characterised by
axonal demyelination and degeneration. Hypothesised as autoimmune, antibodies to neuronal
and myelin proteins have been reported, but there is little evidence of pathogenic antibodies
against cell membrane targets. The aim of this thesis was to investigate whether MS patient sera
contain antibodies against neuronal or oligodendroglial cells, and to develop techniques by which
protein antigenic targets on cell membranes could be identified.
Forty-one MS sera were tested for IgG binding to rat brain. Antibody binding to whlte matter
was more frequent amongst relapsing-remitting MS patients compared to healthy controls, but
western immunoblotting of myelin and rat brain extracts failed to demonstrate any specific
reactivity of MS sera. One hundred MS sera were tested by immunofluorescence for IgG or IgM
binding to the cell surface of neuroblastoma and oligodendroglioma cells. The number of sera
containing cell-binding antibodies was not significantly greater amongst MS patients than
amongst healthy controls.
Using muscle specific tyrosine kinase (MuSK) seropositive myasthenia gravis as an experimental
paradigm, an immunoprecipitation technique to isolate cell surface antigenic targets of serum
antibodies, employing cell surface biotin labelling, was developed. Immunoprecipitated proteins
were visualised with great sensitivity after gel electrophoresis and blotting. Two MS sera were
found 'which specifically immunoprecipitated certain proteins, but development of an unbiased
mass spectrometry technique for identifying immunoprecipitated proteins proved difficult due to
immunoprecipitate impurity.
Eventually, a new protocol for immunoprecipitate digestion and purification prior to mass
spectrometry allowed reliable identification of MuSK exclusively in immunoprecipitates derived
from MuSK seropositive patients. Thus, a clinically relevant cell membrane antigen has been
identi?ed using a novel, non-candidate, unbiased proteomic approach. The same versatile
techruque could be applied to identify proteins in immunoprecipitates derived from patients with
MS or other putative antibody-mediated diseases.
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has been observed on carbon nanotubes than graphite. The adsorption isotherms follow Langmuir
isotherm except cytosine. Mathematical adsorption models devised here reveal that adenine
binding onto graphite could be explained by the interaction between neutral base molecules and
neutral surfaces. The adsorption is suggested to be goyemed by three factors: solubility, basestacking
and the 1t-1t electron interactions.
The adsorption onto quartz, gibbsite and feldspar show little or no adsorption but adsorption onto
'halloysite' is strong with the molecules containing cytosine or adenine groups, especially
cytosine groups. Nucleotides adsorb the most followed by bases and nucleosides in this case. pH
dependence of cytosine adsorption onto halloysite has been analysed by mathematical adsorption
models as well, which shows that the whole adsorption constituents two types of adsorption.
Type I can be ascribed to the binding between =SiO- and protonated cytosine, or the binding
between =SiOH and neutral cytosine independently. Type 2 can be designated to the binding
between =SiO- and neutral cytosine. Mathematical model analysis of literature data illustrates
that the adsorption ofbases/nucleosides onto montmorillonite could be described by two types of
bindings independently: =AIOH groups bind protonated bases/nucleosides, and =AIOH2+ groups
bind neutral bases/nucleosides.-
Graphite could prefer to concentrate nucleosides while halloysite could favour nucleotides
accumulation. Carbon nanotubes and halloysite, which are both tubular, could have facilitated
biomolecules adsorption from diluted primitive oceans. Although no new ideas have been
discovered in the process of origins of life, the adsorption difference onto different minerals have
shown the importance of minerals in concentrating, selecdng and possible protecting and storing
prebiotic biomolecules.
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