Role of Helical Structure in MBP Immunodominant Peptides for Efficient IgM Antibody Recognition in Multiple Sclerosis.

The involvement of Myelin Basic Protein (MBP) in Multiple Sclerosis (MS) has been widely discussed in the literature. This intrinsically disordered protein has an interesting α-helix motif, which can be considered as a conformational epitope. In this work we investigate the importance of the helical structure in antibody recognition by MBP peptides of different lengths. Firstly, we synthesized the peptide MBP (81-106) (1) and observed that its elongation at both N- and C-termini, to obtain the peptide MBP (76-116) (2) improves IgM antibody recognition in SP-ELISA, but destabilizes the helical structure. Conversely, in competitive ELISA, MBP (81-106) (1) is recognized more efficiently by IgM antibodies than MBP (76-116) (2), possibly thanks to its more stable helical structure observed in CD and NMR conformational experiments. These results are discussed in terms of different performances of peptide antigens in the two ELISA formats tested.

Neuroprotective Effect of Glatiramer Acetate on Neurofilament Light Chain Leakage and Glutamate Excess in an Animal Model of Multiple Sclerosis.

Axonal and neuronal pathologies are a central constituent of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE), induced by the myelin oligodendrocyte glycoprotein (MOG) 35-55 peptide. In this study, we investigated neurodegenerative manifestations in chronic MOG 35-55 induced EAE and the effect of glatiramer acetate (GA) treatment on these manifestations. We report that the neuronal loss seen in this model is not attributed to apoptotic neuronal cell death. In EAE-affected mice, axonal damage prevails from the early disease phase, as revealed by analysis of neurofilament light (NFL) leakage into the sera along the disease duration, as well as by immunohistological examination. Elevation of interstitial glutamate concentrations measured in the cerebrospinal fluid (CSF) implies that glutamate excess plays a role in the damage processes inflicted by this disease. GA applied as a therapeutic regimen to mice with apparent clinical symptoms significantly reduces the pathological manifestations, namely apoptotic cell death, NFL leakage, histological tissue damage, and glutamate excess, thus corroborating the neuroprotective consequences of this treatment.

Astrocytes in Multiple Sclerosis-Essential Constituents with Diverse Multifaceted Functions.

In multiple sclerosis (MS), astrocytes respond to the inflammatory stimulation with an early robust process of morphological, transcriptional, biochemical, and functional remodeling. Recent studies utilizing novel technologies in samples from MS patients, and in an animal model of MS, experimental autoimmune encephalomyelitis (EAE), exposed the detrimental and the beneficial, in part contradictory, functions of this heterogeneous cell population. In this review, we summarize the various roles of astrocytes in recruiting immune cells to lesion sites, engendering the inflammatory loop, and inflicting tissue damage. The roles of astrocytes in suppressing excessive inflammation and promoting neuroprotection and repair processes is also discussed. The pivotal roles played by astrocytes make them an attractive therapeutic target. Improved understanding of astrocyte function and diversity, and the mechanisms by which they are regulated may lead to the development of novel approaches to selectively block astrocytic detrimental responses and/or enhance their protective properties.

The survival and function of IL-10-producing regulatory B cells are negatively controlled by SLAMF5.

B cells have essential functions in multiple sclerosis and in its mouse model, experimental autoimmune encephalomyelitis, both as drivers and suppressors of the disease. The suppressive effects are driven by a regulatory B cell (Breg) population that functions, primarily but not exclusively, via the production of IL-10. However, the mechanisms modulating IL-10-producing Breg abundance are poorly understood. Here we identify SLAMF5 for controlling IL-10+ Breg maintenance and function. In EAE, the deficiency of SLAMF5 in B cells causes accumulation of IL10+ Bregs in the central nervous system and periphery. Blocking SLAMF5 in vitro induces both human and mouse IL-10-producing Breg cells and increases their survival with a concomitant increase of a transcription factor, c-Maf. Finally, in vivo SLAMF5 blocking in EAE elevates IL-10+ Breg levels and ameliorates disease severity. Our results suggest that SLAMF5 is a negative moderator of IL-10+ Breg cells, and may serve as a therapeutic target in MS and other autoimmune diseases.

Titration of myelin oligodendrocyte glycoprotein (MOG) - Induced experimental autoimmune encephalomyelitis (EAE) model.

BACKGROUND: Experimental autoimmune encephalomyelitis (EAE) induced by the myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, is a widely used multiple sclerosis (MS) model. Unlike the spontaneous occurrence of MS, in EAE, external immunization with the MOG peptide (200-300 µg/mouse), emulsified in adjuvant enriched with Mycobacterium Tuberculosis (MT) H37Ra (100-500 µg mouse), and pertussis toxin (PTx, 200-500 ng/mouse) injections, are applied, which heavily boosts the immune system. NEW METHOD: A detailed and systematic titration of the MOG 35-55 EAE induction protocol in C57BL/6 mice reveals the minimal doses of the MOG 35-55 peptide, MT H37Ra, and PTx, required for disease manifestation. RESULTS: The amounts of MOG 35-55 peptide, MT H37Ra, and PTx can be drastically reduced from the standard protocol, to level of 5 µg MOG, 25 µg MT H37Ra, and 50 ng PTx, without affecting the clinical manifestations. The titrated protocols induced a high disease incidence and a consistent robust disease course, with full histopathological characteristics of the MOG model, inflammation, demyelination and axonal damage. COMPARISON WITH EXISTING METHODS: Similar disease incidences, day of symptoms appearance, maximal clinical score, and histopathology were obtained for the standard and the titrated protocols. CONCLUSIONS: Reducing the reagent dosages used for EAE induction, without attenuating the disease, can give a truer and less artificial perspective of MS. We propose an improved protocol for this extensively used model, with high disease incidence, a consistent robust course, and characteristic histological manifestations, which may be more sensitive for testing therapeutic modalities, cost-effective, and less distressing to the animals.

Glatiramer acetate increases T- and B -regulatory cells and decreases granulocyte-macrophage colony-stimulating factor (GM-CSF) in an animal model of multiple sclerosis.

To identify the mechanisms relevant for the therapeutic effect of glatiramer acetate (GA), we studied T- and B- regulatory cells as well as GM-CSF expression in mice recovered from experimental autoimmune encephalomyelitis (EAE). Selective depletion of Tregs reduced but did not eliminate the ability of GA to ameliorate EAE, indicating a role for additional immune-subsets. The prevalence of Bregs in the periphery and the CNS of EAE-mice increased following GA-treatment. Furthermore, GA downregulated the pathological expression of GM-CSF, on both the protein and mRNA levels. These findings corroborate the broad immunomodulatory mechanism of action of GA in EAE/MS.

Tuftsin-phosphorylcholine attenuate experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) which carries a significant burden of morbidity and mortality. Herein we examine the effects of acute treatment with tuftsin-phosphorylcholine (TPC), a novel immune-modulating helminth derived compound, on a murine model of MS. Experimental autoimmune encephalomyelitis (EAE) mice received acute treatment with TPC showed an improved clinical score and significantly less signs of inflammation and demyelination in CNS tissue compared with vehicle treated EAE mice. Our findings suggest that TPC may provide a beneficial clinical effect in EAE and may therefore have a potential value for ameliorating clinical manifestations and delaying disease progression in MS.

Cognitive impairment in an animal model of multiple sclerosis and its amelioration by glatiramer acetate.

The severe motor impairment in the MS animal model experimental autoimmune encephalomyelitis (EAE) obstructs the assessment of cognitive functions. We developed an experimental system that evaluates memory faculties in EAE-affected mice, irrespective of their motor performance, enabling the assessment of cognitive impairments along the disease duration, the associated brain damage, and the consequences of glatiramer acetate (GA) treatment on these manifestations. The delayed-non-matching to sample (DNMS) T-maze task, testing working and long term memory was adapted and utilized. Following the appearance of clinical manifestations task performances of the EAE-untreated mice drastically declined. Cognitive impairments were associated with disease severity, as indicated by a significant correlation between the T-maze performance and the clinical symptoms in EAE-untreated mice. GA-treatment conserved cognitive functions, so that despite their exhibited mild motor impairments, the treated mice performed similarly to naïve controls. The cognitive deficit of EAE-mice coincided with inflammatory and neurodegenerative damage to the frontal cortex and the hippocampus; these damages were alleviated by GA-treatment. These combined findings indicate that in addition to motor impairment, EAE leads to substantial impairment of cognitive functions, starting at the early stages and increasing with disease aggravation. GA-treatment, conserves cognitive capacities and prevents its disease related deterioration.

Glatiramer Acetate: from Bench to Bed and Back.

BACKGROUND: Glatiramer acetate (GA, Copaxone®, Copolymer1, Cop 1) is an approved drug for the treatment of relapsing-remitting multiple sclerosis (RRMS). Its efficacy in reducing the frequency of exacerbations and its safety profile establish it as a first-line therapy for MS. Evidence from the animal model experimental autoimmune encephalomyelitis (EAE) and from MS patients indicate that GA affects various levels of the innate and the adaptive immune response, inducing deviation from the pro-inflammatory to the anti-inflammatory pathways. This includes mainly the induction of Th2/3 and T-regulatory cells, and down-regulation of both Th1 and Th17 cells. The immune cells induced by GA reach the CNS and secrete in situ anti-inflammatory cytokines, alleviating the pathological processes. In addition to its immunomodulatory activities, GA promotes neuroprotective repair processes such as secretion of neurotrophic factors, remyelination and neurogenesis, indicating that repair process in the CNS can be up-regulated by therapy.

Astrocyte disruption of neurovascular communication is linked to cortical damage in an animal model of multiple sclerosis.

To elucidate mechanisms contributing to cortical pathology in multiple sclerosis (MS), we investigated neurovascular aberrations, in particular the association of astrocytes with cortical neurons and blood vessels, in mice induced with experimental autoimmune encephalomyelitis (EAE). Blood-brain barrier (BBB) dysfunction was evident by leakage of the tracer sodium fluorescein, along with reduced expression of claudin-5 by endothelial cells and desmin by pericytes. Immunohistological and ultrastructural analyses revealed detachment of the astroglial cell bodies from the blood vessels and loss of their connections with both the blood vessels and the neuronal synapses. Furthermore, examination of individual astrocytic processes at cortical layer IV, where well-defined neuronal columns (barrels) are linked to functional properties, revealed loss of astrocytic confinement to the functional neuronal boundaries. Thus, in contrast to the highly modulated patches of astrocyte processes in naïve mice overlapping the barrel cores, in EAE-mice process distribution was uniform ignoring the barrel boundaries. These aberrations are attributed to the surrounding inflammation, indicated by T-cells presence in the cortex as well as in the subcortical white matter and the meninges. Immunomodulatory treatment with glatiramer acetate partially abrogated the neurovascular damage. These combined findings indicate that under inflammatory conditions, activated perivascular astrocytes fail in neuro-hemodynamic coupling, resulting in obstructed cross-talk between the blood vessels and the neurons. We propose that loss of cortical astrocytic regulation and fine-tuning between the blood supply and the neuronal needs contributes to the neurological impairment and cognitive decline occurring in EAE/MS as well as to the disease progression.

Antibodies from multiple sclerosis patients preferentially recognize hyperglucosylated adhesin of non-typeable Haemophilus influenzae.

In autoimmune diseases, there have been proposals that exogenous "molecular triggers", i.e., specific 'non-self antigens' accompanying infectious agents, might disrupt control of the adaptive immune system resulting in serious pathologies. The etiology of multiple sclerosis (MS) remains unclear. However, epidemiologic data suggest that exposure to infectious agents may be associated with increased MS risk and progression may be linked to exogenous, bacterially-derived, antigenic molecules, mimicking mammalian cell surface glycoconjugates triggering autoimmune responses. Previously, antibodies specific to a gluco-asparagine (N-Glc) glycopeptide, CSF114(N-Glc), were identified in sera of an MS patient subpopulation. Since the human glycoproteome repertoire lacks this uniquely modified amino acid, we turned our attention to bacteria, i.e., Haemophilus influenzae, expressing cell-surface adhesins including N-Glc, to establish a connection between H. influenzae infection and MS. We exploited the biosynthetic machinery from the opportunistic pathogen H. influenzae (and the homologous enzymes from A. pleuropneumoniae) to produce a unique set of defined glucosylated adhesin proteins. Interestingly we revealed that a hyperglucosylated protein domain, based on the cell-surface adhesin HMW1A, is preferentially recognized by antibodies from sera of an MS patient subpopulation. In conclusion the hyperglucosylated adhesin is the first example of an N-glucosylated native antigen that can be considered a relevant candidate for triggering pathogenic antibodies in MS.

Assessing remyelination - metabolic labeling of myelin in an animal model of multiple sclerosis.

The lack of biomarkers is a major obstacle for investigating myelin repair. We used metabolic incorporation of the choline analog - propargyl-choline (P-Cho) to label and visualize newly synthesized myelin in the CNS of mice induced with experimental autoimmune encephalomyelitis (EAE). We further developed unbiased colocalization analysis to quantify P-Cho incorporation specifically into the myelin. Our findings indicate that P-Cho injection to mice recovering from EAE, either spontaneously or following glatiramer acetate treatment, results in significant elevation of its incorporation into the myelin, offering a novel strategy for assessing remyelination in animal models and the remyelination potential of candidate drugs.

Structural Transition in Myelin Membrane as Initiator of Multiple Sclerosis.

In demyelinating diseases such as multiple sclerosis, disrupted myelin structures impair the functional role of the sheath as an insulating layer for proper nerve conduction. Though the etiology and recovery pathways remain unclear, in vivo studies show alterations in the lipid and the adhesive protein (myelin basic protein, MBP) composition. We find that in vitro cytoplasmic myelin membranes with modified lipid composition and low MBP concentration, as in demyelinating disease, show structural instabilities and pathological phase transition from a lamellar to inverted hexagonal, which involve enhanced local curvature. Similar curvatures are also found in vivo in diseased myelin sheaths. In addition, MBP dimers form a correlated mesh-like network within the inner membrane space, only in the vicinity of native lipid composition. These findings delineate the distinct functional roles of dominant constituents in cytoplasmic myelin sheaths, and shed new light on mechanisms disrupting lipid-protein complexes in the diseased state.

Astrocyte morphology is confined by cortical functional boundaries in mammals ranging from mice to human.

Cortical blood flow can be modulated by local activity across a range of species; from barrel-specific blood flow in the rodent somatosensory cortex to the human cortex, where BOLD-fMRI reveals numerous functional borders. However, it appears that the distribution of blood capillaries largely ignores these functional boundaries. Here we report that, by contrast, astrocytes, a major player in blood-flow control, show a striking morphological sensitivity to functional borders. Specifically, we show that astrocyte processes are structurally confined by barrel boundaries in the mouse, by the border of primary auditory cortex in the rat and by layers IIIa/b and Cytochrome Oxidase (CO)-blobs boundaries in the human primary visual cortex. Thus, astrocytes which are critical elements in neuro-hemodynamic coupling show a significant anatomical segregation along functional boundaries across different mammalian species. These results may open a new anatomical marker for delineating functional borders across species, including post-mortem human brains.

Epitope mapping of anti-myelin oligodendrocyte glycoprotein (MOG) antibodies in a mouse model of multiple sclerosis: microwave-assisted synthesis of the peptide antigens and ELISA screening.

The role of pathologic auto-antibodies against myelin oligodendrocyte glycoprotein (MOG) in multiple sclerosis is a highly controversial matter. As the use of animal models may enable to unravel the molecular mechanisms of the human disorder, numerous studies on multiple sclerosis are carried out using experimental autoimmune encephalomyelitis (EAE). In particular, the most extensively used EAE model is obtained by immunizing C57BL/6 mice with the immunodominant peptide MOG(35-55). In this scenario, we analyzed the anti-MOG antibody response in this model using the recombinant refolded extracellular domain of the protein, MOG(1-117). To assess the presence of a B-cell intramolecular epitope spreading mechanism, we tested also five synthetic peptides mapping the 1-117 sequence of MOG, including MOG(35-55). For this purpose, we cloned, expressed in Escherichia coli and on-column refolded MOG(1-117), and we applied an optimized microwave-assisted solid-phase synthetic strategy to obtain the designed peptide sequences. Subsequently, we set up a solid-phase immunoenzymatic assay testing both naïve and EAE mice sera and using MOG protein and peptides as antigenic probes. The results obtained disclose an intense IgG antibody response against both the recombinant protein and the immunizing peptide, while no response was observed against the other synthetic fragments, thus excluding the presence of an intramolecular epitope spreading mechanism. Furthermore, as the properly refolded recombinant probe is able to bind antibodies with greater efficiency compared with MOG(35-55), we hypothesize the presence of both linear and conformational epitopes on MOG(35-55) sequence.

Remyelination in multiple sclerosis: realizing a long-standing challenge.

Multiple sclerosis (MS) is a multifaceted disease, in which an inflammatory autoimmune attack on the myelin in the central nervous system (CNS) leads to extensive demyelination and subsequent axonal pathology. The challenge for MS therapy is to combine effective immunomodulatory therapies with novel neuroprotective approaches that promote repair, in particular remyelination, beyond its limited spontaneous extent. Cumulative findings indicate that immunomodulatory treatments can induce neuroprotective outcomes and provide a supportive milieu for repair processes. Growing understanding of MS pathology together with biotechnological advances has resulted in promising strategies such as inhibitory molecules, monoclonal antibodies and cell therapies. Several candidates that have shown significant effects on the oligodendrocyte population and/or myelin synthesis in animal models are currently or soon to be tested in clinical trials.

Perforin-Positive Dendritic Cells Exhibit an Immuno-regulatory Role in Metabolic Syndrome and Autoimmunity.

Emerging evidence suggests that immunological mechanisms underlie metabolic control of adipose tissue. Here, we have shown the regulatory impact of a rare subpopulation of dendritic cells, rich in perforin-containing granules (perf-DCs). Using bone marrow transplantation to generate animals selectively lacking perf-DCs, we found that these chimeras progressively gained weight and exhibited features of metabolic syndrome. This phenotype was associated with an altered repertoire of T cells residing in adipose tissue and could be completely prevented by T cell depletion in vivo. A similar impact of perf-DCs on inflammatory T cells was also found in a well-defined model of multiple sclerosis, experimental autoimmune encephlalomyelitis (EAE). Thus, perf-DCs probably represent a regulatory cell subpopulation critical for protection from metabolic syndrome and autoimmunity.