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b'This study investigated the immunobiology of MOG-induced EAE in the DA rat, an \\nanimal model, which reproduces the immunopathology of the type II MS lesion (Lucchinetti et \\nal., 2000). A newly established immunisation protocol results in a highly synchronised biphasic \\nform of EAE, which mimics the disease course of secondary progressive MS, albeit in a \\nstrongly abbreviated time course (Figure 3.1.1). This study demonstrates that MOG-specific \\nautoantibodies are responsible for initiating clinical relapse and driving disease progression. On \\nthe background of mild, sub-clinical inflammatory activity in the CNS, pathogenic antibodies \\nenter the CNS and mediate demyelination, a process that in turn amplifies the local \\ninflammatory response (Figure 3.1.14 A). It should however be noted that lethal clinical \\nrelapses may also occur in the absence of a pathogenic antibody response if an inflammatory \\nlesion develops in a region of the CNS that is particularly sensitive to damage, or where it may \\nperturb vital functions, such as the brain stem. Although antibodies have been shown to amplify \\nthe severity of ongoing clinical EAE (Schluesener et al., 1987; Linington et al., 1988; Lassmann \\net al., 1988), firm evidence for a role in driving relapse and disease progression was missing. \\nThis study has now established this principal, which in all probability is relevant to our \\nunderstanding of the pathogenesis of severe, steroid non-responsive relapses in MS patients. \\nHowever, this model of EAE is an artificial system, in which the role of antibody is only \\napparent because of the different kinetics of MOG-specific T and B cell responses. In MS we \\nstill have to answer two crucial questions, namely the identity of the autoantigens targeted by \\nthe demyelinating antibody response, and the factors that may trigger this response. \\nMOG is the only myelin protein known to initiate a demyelinating antibody response in \\nEAE, and MOG-induced EAE has provided a valuable tool to identify the role of pathogenic \\nautoantibodies in immune mediated demyelination. However, there is a major discrepancy \\nbetween the proportion of MS patients with pathogenic MOG-specific antibodies in their \\ncirculation (5%; Haase et al., 2000) and the frequency of patients with pathological changes \\nsuggestive of antibody-mediated pathomechanisms (>50%; Lucchinetti et al., 2000). This \\ndiscrepancy may in part be accounted for by the absorption of the pathogenic antibodies into the \\nCNS, which will lead to a dramatic reduction of the antibody titre in the periphery, as \\ndemonstrated in section 3.1.3.4 of this study. On the other hand, it is unlikely that MOG is the \\nonly target autoantigen, which is exposed on the myelin surface and can therefore initiate a \\ndemyelinating autoantibody response. The identification of potential targets is a prerequisite to develop diagnostic kits to identify those patients with pathogenic autoantibody responses and \\nthen provide an appropriate therapy such as plasma exchange, or immuno-absorption. \\nAs demonstrated in this study, DNA vaccination using a plasmid encoding a myelin \\nantigen is one approach to generate high titre autoantibody responses directed against the native \\nprotein. The pathogenicity of this antibody response can then be assayed in the same animal by \\ninducing EAE. This method circumvents problems such as purity, yield and denaturation, all of \\nwhich complicate any study using antigens isolated from the CNS or generated using \\nrecombinant technologies. Coupling this approach to a proteomics based analysis of the myelin \\nmembrane and reverse genomics to identify candidate gene products provides the means to map \\nout those protein antigens that can be targeted by a demyelinating autoantibody response. The \\nfeasibility of this concept is currently being tested in the rat using PLP and MAG as myelin \\ncomponents that may in certain circumstances provoke a pathogenic autoantibody response. \\nSuch an analysis will, however, not detect pathogenic antibody responses to glycolipid \\nantigens, which are major target autoantigens in a number of diseases affecting the peripheral \\nnervous system such as Guillain Barr\\xe9 syndrome (GBS). In GBS a pathogenic antibody \\nresponse to gangliosides appears to be triggered by infections with particular serotypes of \\nCampylobacter jejuni (Fredman, 1998; Willison and O\\xb4Hanlon, 1999). In the majority of \\npatients these antibody responses are an acute phenomenon and disappear as the patients \\nrecover (Hahn, 1998). It is conceivable that a similar mechanism is responsible for the initiation \\nof severe relapses in some MS patients, if an infection triggers a cross-reactive antibody \\nresponse to a surface glycolipid epitope. This would induce an episode of acute CNS \\ndemyelination that would not be immediately responsive to immunosuppressive therapy, as \\ntissue damage and amplification of the local inflammatory response would be driven by the pre-\\nexisting antibody response. Analysis of the autoantibody responses in MS should therefore be \\nextended to examine lipid as well as protein autoantigens. Such studies should also not be \\nrestricted to myelin, but also address the question of responses to other structures such as the \\naxon and oligodendrocyte progenitor cells. \\nSuch autoantibody responses are however only conditionally pathogenic, in other words \\ntheir pathogenic potential is only expressed if they can enter the CNS across the blood brain \\nbarrier (BBB)(Litzenburger et al., 1998; Bourquin et al., 2000). In EAE the inflammatory insult \\nto the CNS is responsible for the disruption of BBB function and the entry of antibody into the \\nnervous system. MS is characterised by repeated episodes of CNS inflammation but what \\ninitiates and maintains this response is unclear. The observation, that DA rats develop a similar, \\nalthough eventually self-limiting response in the CNS after immunisation with MOG-peptide in CFA provides a model to investigate the immuno-regulatory deficit(s) responsible for chronic \\nCNS inflammation. The disease model is very reproducible with >90% of animals relapsing \\nafter peptide immunisation as opposed to <40% after immunisation with MBP in IFA \\n(Lorentzen et al., 1995). This will make it feasible to use genetic methods, such as disease \\ninduction in congenic and intra-MHC congenic rat strains and whole genome screens in F2 \\nbackcrosses, to identify genetic loci responsible for this defect. The understanding of the \\nmechanisms involved may help to identify new targets for therapeutic strategies concerning \\nchronic inflammatory diseases like MS and rheumatoid arthritis (RA). \\nThe second part of this thesis investigated a very different aspect of the autoimmune \\nresponse to MOG, the consequences of immunological cross-reactivity with BTN, a major \\ncomponent of the milk fat globule membrane. The demonstration of cross-reactive T cell \\nresponses between MOG and a dietary antigen opens a new perspective for the aetiology of MS, \\nsince former investigations of environmental influences were concentrated on molecular \\nmimicry with microbial peptides (Bray et al., 1983; Wucherpfennig et al., 1995; Challoner et \\nal., 1995; Gautam et al., 1998; Ufret-Vicency et al., 1998; Burgoon et al., 1999). \\nEpidemiological studies identified a link between milk consumption and other dietary factors \\nand MS (Butcher, 1986; Malosse et al., 1992; Lauer, 1997), but the identity of the mechanistic \\nbasis in the immune system was unknown. It would be na\\xefve to imagine that milk in the diet \\nwould per se induce an auto-aggressive response to MOG and thereby trigger MS. Indeed, \\ndisease induction is now thought to involve the chance interactions of several environmental \\nfactors on a susceptible genotype. In the case of the cross-reactive pair of antigens BTN/MOG, \\nBTN in the diet would normally induce oral tolerance to the cross-reactive epitope, but this may \\nbe broken either by early post-natal exposure to bovine milk products (Miller et al., 1994), \\ngastro-intestinal infections (Hornquist and Lycke, 1993; Weiner 1997) or a combination of both \\n(as discussed in Introduction 3.2). However, would this combination of effects be sufficient to \\ninduce an inflammatory autoimmune mediated response in the CNS? \\nAny prediction would at this time be premature. Analysis in the DA rat revealed that the \\ncross-reactive repertoire in this model is complex and involves multiple clonal expansions. \\nMoreover, the sequence of the BTN peptide is not identical to the corresponding MOG \\nsequence and the cross-reactive BTN peptide acts as an APL. In view of the degeneracy of TCR \\n- peptide/MHC recognition, the BTN peptide may initiate a range of responses ranging from \\nsuperagonistic (Vergelli et al., 1997) to antagonistic (DeMagistris et al., 1992) in the different T \\ncell clones. The identification of TCR -chains used by the cross-reactive T cells is a first step \\ntowards generating a transgenic animal model. This may allow us to examine the immunopathological consequences of cross-reactivity involving a dietary antigen and the \\nimpact of manipulating the gastro-intestinal flora on the immune response. \\nWhether or not this is relevant for the aetiology of MS is uncertain. Certainly T cell cross-\\nreactivity between the two proteins is very limited and as yet was only demonstrated in the \\ncontext of the RT1av1 rat MHC haplotype (Stefferl et al., 2000). No cross-reactive T cell \\nresponse was detected in LEW and BN rats (Stefferl et al., 2000), as well as in SJL/J, C57/BL6, \\nDBA.1 and CBA.1 mice (Schubart and Wissing, unpublished results). In addition, despite the \\npresence of regions with a high level of sequence identity between the two proteins it was not \\npossible to induce a significant cross-reactive antibody response in the DA rat (section 3.2.7). \\nWhy is this? \\nIn the course of this study it became apparent that BTN is a member of a family of \\nstructurally related gene products that are termed the BTN-, or extended B7- gene family. The \\nN-terminal IgV-like domain of all these proteins exhibits a high degree of amino acid sequence \\nidentity with both MOG and BTN (Henry et al., 1999; Stammers et al., 2000; Rhodes et al., \\n2001; see discussion of chapter 3.2), and members of this family are expressed in a variety of \\norgans. It is possible that during B and T cell maturation in the bone marrow and thymus, cross-\\nreactive peptides derived from these proteins will eliminate many clones that would otherwise \\ncross-react with MOG reactive T and B cells, and only those cells which escape this network of \\ntolerogenic stimuli enter the periphery. \\nAs the sequences of the BTN-gene family members in the rat are currently unknown \\n(with the exception of MOG), the identification of the extracellular domains of rat BTN \\nprovides the first opportunity to test this hypothesis. The oral consumption of milk during \\nsuckling should induce rat-BTN-specific suppressor T cells, characterised by low proliferative \\nresponsiveness and the secretion of IL-10 and TGF- (Weiner et al., 1994). The identification \\nand characterisation (epitope specificity) of these suppressor T cell responses in the different rat \\nstrains (LEW, BN and DA) may provide an explanation why MOG-specific T cell responses are \\nonly poorly encephalitogenic in Lewis rats and might help to elucidate the mechanisms of the \\ndevelopment of tolerance in the newborn animals.'