Aspects of the Immunobiology of Myelin Oligodendrocyte Glycoprotein (MOG)-induced Experimental Autoimmune Encephalomyelitis (EAE)

This study investigated the immunobiology of MOG-induced EAE in the DA rat, an animal model, which reproduces the immunopathology of the type II MS lesion (Lucchinetti et al., 2000). A newly established immunisation protocol results in a highly synchronised biphasic form of EAE, which mimics the disease course of secondary progressive MS, albeit in a strongly abbreviated time course (Figure 3.1.1). This study demonstrates that MOG-specific autoantibodies are responsible for initiating clinical relapse and driving disease progression. On the background of mild, sub-clinical inflammatory activity in the CNS, pathogenic antibodies enter the CNS and mediate demyelination, a process that in turn amplifies the local inflammatory response (Figure 3.1.14 A). It should however be noted that lethal clinical relapses may also occur in the absence of a pathogenic antibody response if an inflammatory lesion develops in a region of the CNS that is particularly sensitive to damage, or where it may perturb vital functions, such as the brain stem. Although antibodies have been shown to amplify the severity of ongoing clinical EAE (Schluesener et al., 1987; Linington et al., 1988; Lassmann et al., 1988), firm evidence for a role in driving relapse and disease progression was missing. This study has now established this principal, which in all probability is relevant to our understanding of the pathogenesis of severe, steroid non-responsive relapses in MS patients. However, this model of EAE is an artificial system, in which the role of antibody is only apparent because of the different kinetics of MOG-specific T and B cell responses. In MS we still have to answer two crucial questions, namely the identity of the autoantigens targeted by the demyelinating antibody response, and the factors that may trigger this response. MOG is the only myelin protein known to initiate a demyelinating antibody response in EAE, and MOG-induced EAE has provided a valuable tool to identify the role of pathogenic autoantibodies in immune mediated demyelination. However, there is a major discrepancy between the proportion of MS patients with pathogenic MOG-specific antibodies in their circulation (5%; Haase et al., 2000) and the frequency of patients with pathological changes suggestive of antibody-mediated pathomechanisms (>50%; Lucchinetti et al., 2000). This discrepancy may in part be accounted for by the absorption of the pathogenic antibodies into the CNS, which will lead to a dramatic reduction of the antibody titre in the periphery, as demonstrated in section 3.1.3.4 of this study. On the other hand, it is unlikely that MOG is the only target autoantigen, which is exposed on the myelin surface and can therefore initiate a demyelinating autoantibody response. The identification of potential targets is a prerequisite to develop diagnostic kits to identify those patients with pathogenic autoantibody responses and then provide an appropriate therapy such as plasma exchange, or immuno-absorption. As demonstrated in this study, DNA vaccination using a plasmid encoding a myelin antigen is one approach to generate high titre autoantibody responses directed against the native protein. The pathogenicity of this antibody response can then be assayed in the same animal by inducing EAE. This method circumvents problems such as purity, yield and denaturation, all of which complicate any study using antigens isolated from the CNS or generated using recombinant technologies. Coupling this approach to a proteomics based analysis of the myelin membrane and reverse genomics to identify candidate gene products provides the means to map out those protein antigens that can be targeted by a demyelinating autoantibody response. The feasibility of this concept is currently being tested in the rat using PLP and MAG as myelin components that may in certain circumstances provoke a pathogenic autoantibody response. Such an analysis will, however, not detect pathogenic antibody responses to glycolipid antigens, which are major target autoantigens in a number of diseases affecting the peripheral nervous system such as Guillain Barré syndrome (GBS). In GBS a pathogenic antibody response to gangliosides appears to be triggered by infections with particular serotypes of Campylobacter jejuni (Fredman, 1998; Willison and O´Hanlon, 1999). In the majority of patients these antibody responses are an acute phenomenon and disappear as the patients recover (Hahn, 1998). It is conceivable that a similar mechanism is responsible for the initiation of severe relapses in some MS patients, if an infection triggers a cross-reactive antibody response to a surface glycolipid epitope. This would induce an episode of acute CNS demyelination that would not be immediately responsive to immunosuppressive therapy, as tissue damage and amplification of the local inflammatory response would be driven by the pre- existing antibody response. Analysis of the autoantibody responses in MS should therefore be extended to examine lipid as well as protein autoantigens. Such studies should also not be restricted to myelin, but also address the question of responses to other structures such as the axon and oligodendrocyte progenitor cells. Such autoantibody responses are however only conditionally pathogenic, in other words their pathogenic potential is only expressed if they can enter the CNS across the blood brain barrier (BBB)(Litzenburger et al., 1998; Bourquin et al., 2000). In EAE the inflammatory insult to the CNS is responsible for the disruption of BBB function and the entry of antibody into the nervous system. MS is characterised by repeated episodes of CNS inflammation but what initiates and maintains this response is unclear. The observation, that DA rats develop a similar, although 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 CNS inflammation. The disease model is very reproducible with >90% of animals relapsing after peptide immunisation as opposed to <40% after immunisation with MBP in IFA (Lorentzen et al., 1995). This will make it feasible to use genetic methods, such as disease induction in congenic and intra-MHC congenic rat strains and whole genome screens in F2 backcrosses, to identify genetic loci responsible for this defect. The understanding of the mechanisms involved may help to identify new targets for therapeutic strategies concerning chronic inflammatory diseases like MS and rheumatoid arthritis (RA). The second part of this thesis investigated a very different aspect of the autoimmune response to MOG, the consequences of immunological cross-reactivity with BTN, a major component of the milk fat globule membrane. The demonstration of cross-reactive T cell responses between MOG and a dietary antigen opens a new perspective for the aetiology of MS, since former investigations of environmental influences were concentrated on molecular mimicry with microbial peptides (Bray et al., 1983; Wucherpfennig et al., 1995; Challoner et al., 1995; Gautam et al., 1998; Ufret-Vicency et al., 1998; Burgoon et al., 1999). Epidemiological studies identified a link between milk consumption and other dietary factors and MS (Butcher, 1986; Malosse et al., 1992; Lauer, 1997), but the identity of the mechanistic basis in the immune system was unknown. It would be naïve to imagine that milk in the diet would per se induce an auto-aggressive response to MOG and thereby trigger MS. Indeed, disease induction is now thought to involve the chance interactions of several environmental factors on a susceptible genotype. In the case of the cross-reactive pair of antigens BTN/MOG, BTN in the diet would normally induce oral tolerance to the cross-reactive epitope, but this may be broken either by early post-natal exposure to bovine milk products (Miller et al., 1994), gastro-intestinal infections (Hornquist and Lycke, 1993; Weiner 1997) or a combination of both (as discussed in Introduction 3.2). However, would this combination of effects be sufficient to induce an inflammatory autoimmune mediated response in the CNS? Any prediction would at this time be premature. Analysis in the DA rat revealed that the cross-reactive repertoire in this model is complex and involves multiple clonal expansions. Moreover, the sequence of the BTN peptide is not identical to the corresponding MOG sequence and the cross-reactive BTN peptide acts as an APL. In view of the degeneracy of TCR - peptide/MHC recognition, the BTN peptide may initiate a range of responses ranging from superagonistic (Vergelli et al., 1997) to antagonistic (DeMagistris et al., 1992) in the different T cell clones. The identification of TCR -chains used by the cross-reactive T cells is a first step towards generating a transgenic animal model. This may allow us to examine the immunopathological consequences of cross-reactivity involving a dietary antigen and the impact of manipulating the gastro-intestinal flora on the immune response. Whether or not this is relevant for the aetiology of MS is uncertain. Certainly T cell cross- reactivity between the two proteins is very limited and as yet was only demonstrated in the context of the RT1av1 rat MHC haplotype (Stefferl et al., 2000). No cross-reactive T cell response was detected in LEW and BN rats (Stefferl et al., 2000), as well as in SJL/J, C57/BL6, DBA.1 and CBA.1 mice (Schubart and Wissing, unpublished results). In addition, despite the presence of regions with a high level of sequence identity between the two proteins it was not possible to induce a significant cross-reactive antibody response in the DA rat (section 3.2.7). Why is this? In the course of this study it became apparent that BTN is a member of a family of structurally related gene products that are termed the BTN-, or extended B7- gene family. The N-terminal IgV-like domain of all these proteins exhibits a high degree of amino acid sequence identity with both MOG and BTN (Henry et al., 1999; Stammers et al., 2000; Rhodes et al., 2001; see discussion of chapter 3.2), and members of this family are expressed in a variety of organs. It is possible that during B and T cell maturation in the bone marrow and thymus, cross- reactive peptides derived from these proteins will eliminate many clones that would otherwise cross-react with MOG reactive T and B cells, and only those cells which escape this network of tolerogenic stimuli enter the periphery. As the sequences of the BTN-gene family members in the rat are currently unknown (with the exception of MOG), the identification of the extracellular domains of rat BTN provides the first opportunity to test this hypothesis. The oral consumption of milk during suckling should induce rat-BTN-specific suppressor T cells, characterised by low proliferative responsiveness and the secretion of IL-10 and TGF- (Weiner et al., 1994). The identification and characterisation (epitope specificity) of these suppressor T cell responses in the different rat strains (LEW, BN and DA) may provide an explanation why MOG-specific T cell responses are only poorly encephalitogenic in Lewis rats and might help to elucidate the mechanisms of the development of tolerance in the newborn animals

Diversity and Distribution of Mites (Acari: Ixodida, Mesostigmata, Trombidiformes, Sarcoptiformes) in the Svalbard Archipelago

Svalbard is a singular region to study biodiversity. Located at a high latitude and geographically isolated, the archipelago possesses widely varying environmental conditions and unique flora and fauna communities. It is also here where particularly rapid environmental changes are occurring, having amongst the fastest increases in mean air temperature in the Arctic. One of the most common and species-rich invertebrate groups in Svalbard is the mites (Acari). We here describe the characteristics of the Svalbard acarofauna, and, as a baseline, an updated inventory of 178 species (one Ixodida, 36 Mesostigmata, 43 Trombidiformes, and 98 Sarcoptiformes) along with their occurrences. In contrast to the Trombidiformes and Sarcoptiformes, which are dominated in Svalbard by species with wide geographical distributions, the Mesostigmata include many Arctic species (39%); it would thus be an interesting future study to determine if mesostigmatid communities are more affected by global warming then other mite groups. A large number of new species (42 spp.) have been described from Svalbard, including 15 that have so far been found exclusively there. It is yet uncertain if any of these latter species are endemic: six are recent findings, the others are old records and, in most cases, impossible to verify. That the Arctic is still insufficiently sampled also limits conclusions concerning endemicity.publishedVersio

Siponimod (BAF312) prevents synaptic neurodegeneration in experimental multiple sclerosis

Data from multiple sclerosis (MS) and the MS rodent model, experimental autoimmune encephalomyelitis (EAE), highlighted an inflammation-dependent synaptopathy at the basis of the neurodegenerative damage causing irreversible disability in these disorders. This synaptopathy is characterized by an imbalance between glutamatergic and GABAergic transmission and has been proposed to be a potential therapeutic target. Siponimod (BAF312), a selective sphingosine 1-phosphate1,5 receptor modulator, is currently under investigation in a clinical trial in secondary progressive MS patients. We investigated whether siponimod, in addition to its peripheral immune modulation, may exert direct neuroprotective effects in the central nervous system (CNS) of mice with chronic progressive EAE

Hypericins as Potential Leads for New Therapeutics

70 years have passed since the first isolation of the naphthodianthrones hypericin and pseudohypericin from Hypericum perforatum L. Today, they continue to be one of the most promising group of polyphenols, as they fascinate with their physical, chemical and important biological properties which derive from their unique chemical structure. Hypericins and their derivatives have been extensively studied mainly for their antitumor, antiviral and antidepressant properties. Notably, hypericin is one of the most potent naturally occurring photodynamic agents. It is able to generate the superoxide anion and a high quantum yield of singlet oxygen that are considered to be primarily responsible for its biological effects. The prooxidant photodynamic properties of hypericin have been exploited for the photodynamic therapy of cancer (PDT), as hypericin, in combination with light, very effectively induces apoptosis and/or necrosis of cancer cells. The mechanism by which these activities are expressed continues to be a main topic of discussion, but according to scientific data, different modes of action (generation of ROS & singlet oxygen species, antiangiogenesis, immune responces) and multiple molecular pathways (intrinsic/extrinsic apoptotic pathway, ERK inhibition) possibly interrelating are implicated. The aim of this review is to analyse the most recent advances (from 2005 and thereof) in the chemistry and biological activities (in vitro and in vivo) of the pure naphthodianthrones, hypericin and pseudohypericin from H. perforatum. Extracts from H. perforatum were not considered, nor pharmakokinetic or clinical data. Computerised literature searches were performed using the Medline (PubMed), ChemSciFinder and Scirus Library databases. No language restrictions were imposed

The next-generation sphingosine 1 receptor modulator BAF312 (siponimod) improves cortical network functionality in autoimmune neurodegeneration

Background. Autoimmune diseases of the central nervous system (CNS) like multiple sclerosis (MS) are characterized by inflammation and demyelinated lesions in white and grey matter regions. While inflammation is present at all stages of MS, it is more pronounced in the relapsing forms of the disease (RMS), whereas progressive MS (PMS) shows significant neuroaxonal damage and grey and white matter atrophy. Hence, disease-modifying treatments beneficial in patients with RMS have limited success in PMS. BAF312 (siponimod) is a novel sphingosine 1-phosphate (S1P) receptor modulator recently shown to delay progression in PMS. It sequesters lymphocytes in the lymphoid tissues, thereby reducing CNS inflammation. Unlike most other immune-modulatory agents, BAF312 crosses the blood-brain barrier (BBB) and binds to S1P receptors on brain-resident cells like neurons, astrocytes and oligodendrocytes. Methods. To evaluate direct neuroprotective effects of BAF312 on neuroaxonal damage, BAF312 was locally administered in the CNS of experimental autoimmune encephalomyelitis (EAE) mice with distinct grey and white matter lesions (focal EAE) using an osmotic mini-pump. Focal lesions were induced by stereotactical injection of pro-inflammatory cytokines in pre-immunized mice. The effects of intracerebral versus systemic BAF312 treatment were compared. Ex vivo flow cytometric assays were performed to investigate the effects on local inflammatory cells. Voltage-sensitive dye imaging was used to investigate the spatio-temporal patterns of neuronal network activity in cortical grey matter. Results. Immune cell infiltration of animals with both grey and white matter lesions was lowered upon systemic administration of BAF312, while intracerebral treatment did not shift the immune phenotype. Examination of neuronal circuits with focal inflammatory lesions revealed an altered activity pattern of activation compared to controls within the cortical layers in response to electrical stimulation of white matter fiber tracts entering the cortex. BAF312 partially restored cortical neuronal circuit function. Conclusion. Intracerebral BAF312 treatment did not influence 1 EAE disease course at the tested doses, irrespectively of the location of focal EAE lesions. However, administration of BAF312 in acute brain slices from focal EAE mice showed improvement of neuronal network functionality. The data suggest that BAF312 exerts a neuroprotective effect after crossing the BBB independently of peripheral effects on immune cells

Kindliche Thränen, Welche bey dem Grabe Des Hoch-Edelgebohrnen, und Hochgelahrten Herrn, Herrn Joahnn Christian Schubarts, Derer Rechte Doctoris, Fürstl. Sachß. Coburg-Meiningischen Hof-Advocati, wie auch E. Hoch- und Wohl-Edlen Stadt-Raths allhier ansehnl. Mitgliedes, Jhres ... werthgeschätztesten Herrn Papa, Als Derselbe Am 4. Decembr. des 1757. Jahres ... entschlieffe, Zu Ausdrückung ihrer äussersten Wehmuth darbringen Vor dessen genossene Väterliche Wohlthaten und Erziehung Aber zugleich ihre schuldigste und aufrichtigste Pflicht Jn nachfolgenden Zeilen darlegen wollten, Dessen HInterlassene beyde Kinder Friederica Bernhardina, Schubartin. Anna Magdalena, [Schubartin]

Vorlageform des Erscheinungsvermerks: Mejnjngen, gedruckt bey Joh. Günther Scheidemanteln, Fürstl. Sächß. Hof-Buchdr

Central nervous system-directed effects of FTY720 (fingolimod).

FTY720, also known as fingolimod, is an orally administered sphingosine-1-phosphate (S1P) analogue that is under investigation as a therapy for both relapsing-remitting (RR) and progressive forms of multiple sclerosis (MS). The demonstrated beneficial effect of FTY720 on disease activity in RR-MS patients and in the animal model experimental autoimmune encephalomyelitis (EAE) is largely attributed to effects on the systemic immune system. In addition, unlike other current systemic immuno-modulators used in MS, the lipophilic nature of FTY720 allows it to cross the blood-brain barrier (BBB). Since S1P receptors are expressed on all cell types, FTY720 has the potential to exert effects directly on the BBB and on resident cells of the CNS. The latter include cells implicated in regulating immune reactivity within the CNS (astrocytes, microglia), those that are targeted by the disease process (oligodendrocytes, neurons), and those involved in repair (oligodendrocyte progenitor cells). In vitro studies document the dose-dependent effects of FTY720 on neural cell survival, differentiation, and cytoskeletal dynamics. Animal model studies, specifically EAE, indicate an overall neuroprotective effect of FTY720 mediated at least in part by its actions within the CNS. Ongoing studies will need to define the direct and indirect (via immune-modulation) effects of FTY720 on the CNS across the broad clinical spectrum of MS

Low-molecular weight inhibitors of the alternative complement pathway.

Dysregulation of the alternative complement pathway predisposes individuals to a number of diseases. It can either be evoked by genetic alterations in or by stabilizing antibodies to important pathway components and typically leads to severe diseases such as paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, C3 glomerulopathy, and age-related macular degeneration. In addition, the alternative pathway may also be involved in many other diseases where its amplifying function for all complement pathways might play a role. To identify specific alternative pathway inhibitors that qualify as therapeutics for these diseases, drug discovery efforts have focused on the two central proteases of the pathway, factor B and factor D. Although drug discovery has been challenging for a number of reasons, potent and selective low-molecular weight (LMW) oral inhibitors have now been discovered for both proteases and several molecules are in clinical development for multiple complement-mediated diseases. While the clinical development of these inhibitors initially focuses on diseases with systemic and/or peripheral tissue complement activation, the availability of LMW inhibitors may also open up the prospect of inhibiting complement in the central nervous system where its activation may also play an important role in several neurodegenerative diseases