Relapsing Experimental Allergic Encephalomyelitis Research Articles

Metallothionein I+II Expression as an Early Sign of Chronic Relapsing Experimental Autoimmune Encephalomyelitis in Rats

Metallothioneins (MTs) are small, cysteine-rich proteins which have been implicated in various forms of stress providing cytoprotective action against oxidative injury, DNA damage and apoptosis. Owing to their high affinity for physiological metals, such as zinc and copper MTs are also critical components of regulatory proteins involved in cell growth and multiplication, as well as in the maintenance of immune homeostasis. To elucidate the role of MTs in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MT I+II proteins and the content of free Zn ions in the brain, spinal cord and in the liver early in the course of chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE) pathogenesis, i.e. before the onset of any clinical symptoms. Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control animals were treated with CFA alone. The data, obtained by immuno-histochemistry and in situ fluorescent labeling of free zinc ions, have shown that in the presymptomatic phase of CR-EAE (on the seventh postimmunization day) MTs I+II were markedly upregulated in the cells that form blood-brain and blood-cerebrospinal fluid barriers, as well as in the cerebellar parenchyma and hippocampal dentate gyri. Furthermore, we found that the liver also becomes a site of extensive MTs I+II synthesis shortly after immunization. Simultaneously, tissue content of free zinc ions increased at the sites of MTs induction, reflecting their antioxidative activity. The data, described in this paper point to regulatory and neuroprotective role of MTs in the pathogenesis of CR-EAE.

B7-H4Ig inhibits mouse and human T-cell function and treats EAE via IL-10/Treg-dependent mechanisms

We evaluated the therapeutic efficacy and mechanisms of action of both mouse and human B7-H4 Immunoglobulin fusion proteins (mB7-H4Ig; hB7-H4Ig) in treating EAE. The present data show that mB7-H4Ig both directly and indirectly (via increasing Treg function) inhibited CD4+ T-cell proliferation and differentiation in both Th1- and Th17-cell promoting conditions while inducing production of IL-10. B7-H4Ig treatment effectively ameliorated progression of both relapsing (R-EAE) and chronic EAE correlating with decreased numbers of activated CD4+ T-cells within the CNS and spleen, and a concurrent increase in number and function of Tregs. The functional requirement for Treg activation in treating EAE was demonstrated by a loss of therapeutic efficacy of hB7-H4Ig in R-EAE following inactivation of Treg function either by anti-CD25 treatment or blockade of IL-10. Significant to the eventual translation of this treatment into clinical practice, hB7-H4Ig similarly inhibited the in vitro differentiation of naïve human CD4+ T-cells in both Th1- and Th17-promoting conditions, while promoting the production of IL-10. B7-H4Ig thus regulates pro-inflammatory T-cell responses by a unique dual mechanism of action and demonstrates significant promise as a therapeutic for autoimmune diseases, including MS.

Serum levels of 25-hydroxy vitamin D in normal Biozzi and C57BL/6 mice and during the course of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE)

The aim of the study was to examine possible variations in the levels of 25-hydroxy vitamin D [25-(OH)D] in sera from normal Biozzi and C57BL/6 mice and during the course of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE). Serum concentrations of 25-(OH)D were measured in normal male and female Biozzi and C57BL/6 mice, at 3-4 weeks old and 8-10 weeks old, with a minimum of six animals/group. Levels of the vitamin were also determined in CR EAE-inoculated mice, and controls, during the course of the disease using a minimum of six animals/treatment. Cardiac blood was collected from the groups of normal, control and CR EAE-sensitised mice and sera prepared, by centrifugation of clotted samples, and assayed for 25-(OH)D levels by chemiluminescence assay. Normal male and female Biozzi and C57BL/6 mice had significantly higher levels of 25-(OH)D at 8-10 weeks old compared to concentrations at 3-4 weeks of age (P < 0.005). Also, levels of the vitamin were significantly raised in C57BL/6 male and female mice compared to values in samples from corresponding Biozzi mice. In addition, the amounts of 25-(OH)D in sera from female Biozzi and C57BL/6 mice were significantly increased compared to strain and aged-matched male mice. The CR EAE mice with acute stage disease had significantly higher 25-(OH)D levels compared to controls (P < 0.005). Vitamin concentrations fell to within controls values with the progression of CR EAE. Our preliminary studies have revealed marked differences between the amounts of 25-(OH)D in sera from Biozzi and C57BL/6 mice together with clear gender bias. The investigations also show significant, but selective changes, in levels of the vitamin during the course of CR EAE that are not always associated with the neurological disease state.

Intraventricularly Injected Olig2-NSCs Attenuate Established Relapsing–Remitting EAE in Mice

In multiple sclerosis (MS), a chronic inflammatory relapsing demyelinating disease, failure to control or repair damage leads to progressive neurological dysfunction and neurodegeneration. Implantation of neural stem cells (NSCs) has been shown to promote repair and functional recovery in the acute experimental autoimmune encephalomyelitis (EAE) animal model for MS; the major therapeutic mechanism of these NSCs appeared to be immune regulation. In the present study, we examined the efficacy of intraventricularly injected NSCs in chronic relapsing experimental autoimmune encephalomyelitis (CREAE), the animal disease model that is widely accepted to mimic most closely recurrent inflammatory demyelination lesions as observed in relapsing-remitting MS. In addition, we assessed whether priming these NSCs to become oligodendrocyte precursor cells (OPCs) by transient overexpression of Olig2 would further promote functional recovery, for example, by contributing to actual remyelination. Upon injection at the onset of the acute phase or the relapse phase of CREAE, NSCs as well as Olig2-NSCs directly migrated toward active lesions in the spinal cord as visualized by in vivo bioluminescence and biofluorescence imaging, and once in the spinal cord, the majority of Olig2-NSCs, in contrast to NSCs, differentiated into OPCs. The survival of Olig2-NSCs was significantly higher than that of injected control NSCs, which remained undifferentiated. Nevertheless, both Olig2-NSCs and NSC significantly reduced the clinical signs of acute and relapsing disease and, in case of Olig2-NSCs, even completely abrogated relapsing disease when administered early after onset of acute disease. We provide the first evidence that NSCs and in particular NSC-derived OPCs (Olig2-NSCs) ameliorate established chronic relapsing EAE in mice. Our experimental data in established neurological disease in mice indicate that such therapy may be effective in relapsing-remitting MS preventing chronic progressive disease.

N‐methyl‐D‐aspartate (NMDA) receptor involvement in central nervous system prostaglandin production during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE)

Our previous studies have established that major changes in central nervous system (CNS) prostaglandin (PG) levels occur during the relapse phase of chronic relapsing experimental autoimmune encephalomyelitis (CR EAE), an animal model of the human demyelinating disease multiple sclerosis. PG production is controlled through a series of enzymic pathways that, in EAE, are influenced by neuroantigen-driven autoimmune events. In non-immune-based models of CNS disease, endogenous glucocorticoids have been proposed as instigators of PG synthesis via activation of the N-methyl-D-aspartate (NMDA) receptor. Glucocorticoids have an important regulatory role in the pathogenesis EAE and the NMDA receptor is intimately involved in many of the characteristic neuroinflammatory processes that govern the disease. Therefore, the alterations in prostanoid concentrations during the relapse stage of CR EAE may ultimately be governed by glucocorticoid-induced NMDA receptor activation. The current investigation has examined the proposed glucocorticoid-NMDA receptor link by determining the effects of the receptor antagonist, (+) MK-801, on CNS PGE 2 and PGD 2 levels in Biozzi mice with relapse symptoms of CR EAE. Prostanoid concentrations in the cerebral cortex were not altered by drug administration, and in cerebellar tissues, a vehicle effect negated any drug-induced changes. However, the level of PGD 2 in spinal cords from (+) MK-801-dosed mice was significantly lower, compared to controls, but PGE 2 concentrations remained unchanged. The results suggest that glucocorticoid-NMDA receptor-linked events are not primarily responsible for PG generation in the brain but may influence prostanoid production in discrete areas of the CNS.

Neuroprotective Effects of Progesterone in Chronic Experimental Autoimmune Encephalomyelitis

Observations so far obtained in experimental autoimmune encephalomyelitis (EAE) have revealed the promising neuroprotective effects exerted by progesterone (PROG). The findings suggest that this neuroactive steroid may potentially represent a therapeutic tool for multiple sclerosis (MS). However, up to now, the efficacy of PROG has been only tested in the acute phase of the disease, whereas it is well known that MS expresses different features depending on the phase of the disease. Accordingly, we have evaluated the effect of PROG treatment in EAE induced in Dark Agouti rats (i.e. an experimental model showing a protracted relapsing EAE). Data obtained 45 days after EAE induction show that PROG treatment exerts a beneficial effect on clinical score, confirming surrogate parameters of spinal cord damage in chronic EAE (i.e. reactive microglia, cytokine levels, activity of the Na(+) ,K(+) -ATPase pump and myelin basic protein expression). An increase of the levels of dihydroprogesterone and isopregnanolone (i.e. two PROG metabolites) was also observed in the spinal cord after PROG treatment. Taken together, these results indicate that PROG is effective in reducing the severity of chronic EAE and, consequently, may have potential with respect to MS treatment.

Durable pharmacological responses from the peptide drug ShK-186, a specific Kv1.3 channel inhibitor that suppresses T cell mediators of autoimmune disease (51.5)

Abstract The Potassium Kv1.3 channel on effector memory T cells is a target to treat autoimmune diseases. ShK-186, a specific peptide inhibitor of Kv1.3, has shown promise in animal models of multiple sclerosis and rheumatoid arthritis. Here, we describe the development of methods to establish a pharmacokinetic (PK) - pharmacodynamic (PD) relationship for ShK-186, and test the hypothesis that the drug has a sustained PD effect in autoimmune disease models. The PK of ShK-186 was evaluated in rats and cynomolgus monkeys using HPLC-MS/MS and ELISA methods to measure the free fraction in plasma. These results were compared with SPECT/CT data collected with an 111In-DOTA-conjugate of ShK to assess whole blood PK parameters as well as the drug’s absorption, distribution and excretion. Our data suggest ShK-186 is absorbed slowly from the injection site resulting in blood concentrations above the Kv1.3 channel-blocking Kd for up to 7 days in monkey. PD studies on human peripheral blood mononuclear cells show that transient ShK-186 treatment results in a sustained suppression of cytokine responses and may contribute to prolonged drug effects. Moreover, in rat delayed-type hypersensitivity and chronic relapsing experimental autoimmune encephalomyelitis, a single dose of drug every 3 - 5 days is as effective as daily administration. These data suggest the PD effects of ShK-186 are governed primarily by slow distribution from peripheral tissues and long residence time on the Kv1.3 channel.

Immunization method influences EAE relapse rate in SJL mice (171.6)

Abstract We have discovered that administration of pertussis toxin as an adjuvant during induction of experimental autoimmune encephalomyelitis (EAE) in SJL mice results in a more severe first EAE wave, but fewer, milder and later relapses than if pertussis toxin is not used. This finding is inconsistent with the widely-accepted epitope spreading hypothesis, which predicts more severe relapses resulting from more severe tissue damage in the first wave. EAE in SJL mice immunized with PLP139-151/CFA is the mouse model which most closely resembles human remitting/relapsing multiple sclerosis. EAE develops in 90-100% of immunized mice 10-15 days after immunization. The first wave of EAE lasts several days and most mice recover from it. After a disease-free period from one day to several months, most mice relapse. 50-75% of mice develop a relapse during the first 6 weeks after immunization. It is not clear why relapses develop. It is widely accepted that relapses result from epitope spreading - new immune responses to epitopes of self antigens other than the PLP139-151. It is believed that tissue damage from the initial immune response allows “presentation” of new epitopes to auto-reactive T cells. Our findings appear to conflict with this hypothesis. Potential mechanisms of relapse suppression by pertussis toxin will be discussed.

Cannabinoids ameliorate disease progression in a model of multiple sclerosis in mice, acting preferentially through CB1 receptor-mediated anti-inflammatory effects

Multiple sclerosis (MS) is an autoimmune disease that affects the CNS and it is characterized by inflammation, demyelination, remyelination, gliosis and axonal damage that occur mainly in the spinal cord. Cannabinoids have been proposed as promising therapeutic agents in MS given their capability to alleviate specific MS symptoms (e.g., spasticity, pain). Although MS has been considered mainly an inflammatory disorder, recent evidence, however, revealed the importance of neurodegenerative events, opening the possibility that cannabinoid agonists, given their cytoprotective properties, may also serve to reduce oligodendrocyte death and axonal damage in MS. Thus, the treatment with WIN55,512-2, a potent CB1 and CB2 agonist, was reported to be effective to ameliorate tremor and spasticity in mice with chronic relapsing experimental autoimmune encephalomyelitis, a murine model of MS, but also to delay disease progression in this and other murine models of MS. The purpose of this investigation was to further explore the mechanism(s) underlying the amelioration in disease progression caused by WIN55,212-2. We have particularly focused on anti-glutamatergic and anti-inflammatory effects of this cannabinoid agonist. In this study, we used mice treated with myelin oligodendrocyte glycoprotein (MOG) that induces a progressive pattern of EAE and conducted the pharmacological experiments in early stages of the disease. As expected, the administration of WIN55,512-2 (5 mg/kg, i.p) had a positive effect in reducing neurological disability and improving motor coordination of EAE mice. Levels of glutamate and GABA in the spinal cord and also in the brainstem of EAE mice were similar to control animals, and, accordingly, they were not altered by the treatment with WIN55,212-2. However, EAE mice showed some subtle alterations in mRNA levels for the glutamate transporter GLT1 and, to a lesser extent, GLAST too, changes that were altered by the treatment with WIN55,212-2 in the spinal cord, but not in the brainstem. Regarding to inflammatory responses, EAE mice showed a marked up-regulation in mRNA levels for COX-2, inducible NOS and TNF-α in the spinal cord and the brainstem, these responses being attenuated after the treatment with WIN55,212-2. We also observed the presence of cell aggregates in the spinal cord of EAE mice that were significantly attenuated by the treatment with WIN55,212-2. Immunohistochemical analysis (with Iba-1 and Cd11b) of these aggregates indicated that they corresponded to microglia (resident macrophages) and peripheral macrophages. Lastly, experiments conducted with selective antagonists for the CB1 (e.g. rimonabant) or CB2 (e.g. AM-630) receptors revealed that WIN55,212-2 effects in EAE mice were mediated by the activation of CB1 but not CB2 receptors, as reflected the reversion of positive effects of this cannabinoid on neurological decline, TNF-α generation and accumulation of cell aggregates in the spinal cord with rimonabant, but not with AM-630. This was concordant with the lack of positive effects on neurological decline observed in EAE mice when they received HU-308, a selective CB2 receptor agonist, instead WIN55,212-2. In summary, the treatment of EAE mice with the cannabinoid agonist WIN55,512-2 reduced their neurological disability and the progression of the disease. This effect was exerted through the activation of CB1 receptors, which would exert a positive influence in the reduction of inflammatory events linked to the pathogenesis of this disease.

Cyclooxygenase expression and prostaglandin levels in central nervous system tissues during the course of chronic relapsing experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS) and its animal counterpart experimental autoimmune encephalomyelitis (EAE) have a major inflammatory component that drives and orchestrates both diseases. One particular group of mediators are the prostaglandins (PGs), which we have previously shown, through quantitation and pharmacological intervention, to be closely involved in the pathology of MS and EAE. The aim of the current study was to determine the expression of the PG-generating cyclooxygenase (COX) enzymes and the profile of PGE(2) and PGD(2), in selected central nervous system (CNS) tissues, with the development of the chronic relapsing (CR) form of EAE. In particular, the work investigates the possible relationship between the expression of COX isoenzymes and PG levels during the neurological phases of CR EAE. CR EAE was induced in Biozzi mice with inoculum containing lyophilised, syngeneic spinal cord emulsified in complete Freund's adjuvant. The cerebral cortex, cerebellum and spinal cord were dissected from mice during the acute, remission and relapse stages of disease with a minimum of five animals per treatment. The expression of COX-1, COX-1b variant and COX-2, in pooled samples, was determined by Western blotting. PGE(2) and PGD(2) levels in extracted samples were measured using commercial enzyme immunoassay kits. COX-2 expression in spinal cords during acute disease remained unaltered and was in contrast to an enhancement of the enzyme, together with COX-1 and COX-1b, in all other sampled areas. PGE(2) and PGD(2) levels remained unchanged during the acute phase and the subsequent remission of symptoms. COX-1 and COX-1b expression was elevated in tissues during the relapse stage of CR EAE and concentrations of the prostanoids were markedly increased. The study examines the implications of COX isoenzyme expression over the course of CR EAE and discusses the reported relationship between PGE(2) and PGD(2) in the instigation and resolution of CNS inflammation. Consideration is also given to the treatment of CR EAE and suggests that drugs designed to limit the inflammatory effects of the PGs should be administered prior to or during the relapse phase of the disease.

PETIR-001, a dual inhibitor of dipeptidyl peptidase IV (DP IV) and aminopeptidase N (APN), ameliorates experimental autoimmune encephalomyelitis in SJL/J mice

Cellular dipeptidyl peptidase IV (DP IV, CD26) and amino-peptidase N (APN, CD13) play regulatory roles in T cell activation and represent potential targets for treatment of inflammatory disorders. We have developed a novel therapeutic strategy, 'peptidase-targeted Immunoregulation' (PETIR™), which simultaneously targets both cellular DP IV and APN via selective binding sites different from the active sites with a single inhibitor. To prove the therapeutic concept of PETIR™ in autoimmunity of the central nervous system (CNS), we evaluated the effect of a single substance, PETIR-001, in an animal model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE) in SJL/J mice. Administration of PETIR-001 significantly delayed and decreased clinical signs of active EAE, when given in a therapeutic manner intraperitoneally from day 15 to day 24 after induction of EAE. Both the acute phase and the first relapse of EAE were markedly inhibited. Importantly, a similar therapeutic benefit was obtained after oral administration of PETIR-001 from day 12 to day 21 after disease induction. Our results demonstrate that PETIR-001 exhibits a therapeutic effect on EAE in SJL/J mice. Thus, PETIR™ represents a novel and efficient therapeutic approach for immunotherapy of CNS inflammation.

Interferon-γ Orchestrates the Number and Function of Th17 Cells in Experimental Autoimmune Encephalomyelitis

Th17 cells are suggested to be pathogenic in mediating experimental autoimmune encephalomyelitis (EAE), but their relation to interferon (IFN)-γ-producing Th1 cells in vivo is not well understood. We studied the numbers and functions of Th17 cells in CREAE in Biozzi ABH mice, both in peripheral lymphoid organs and in the central nervous system. Th1 and Th17 cells alternated in secondary lymphoid organs and infiltrated into the central nervous system during chronic relapsing experimental autoimmune encephalomyelitis (CREAE). In the absence of IFN-γ the numbers and secretion of Th17 cells was enhanced, whereas exogenous administration of IFN-γ decreased the Th17 cells. In mice with intact IFN-γ genes, in vivo neutralization of interleukin (IL)-17 protected against EAE development by enhancing the number of IFN-γ-producing cells. IFN-γ knockout mice were partially protected by anti-IL-17 antibodies by decreasing cell numbers and production of IL-17. Our findings suggest that, whereas IFN-γ as such is not necessary for EAE development in the mouse, the lack of suppression of Th17 cells by IFN-γ enhances the susceptibility to develop EAE. IFN-γ thus orchestrates the number and function of Th17 cells.

PARP-1 inhibition prevents CNS migration of dendritic cells during EAE, suppressing the encephalitogenic response and relapse severity

Background: Pharmacological inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) are currently evaluated in clinical trials for various malignancies but, interestingly, also proved of remarkable efficacy in preclinical models of autoimmune disorders including experimental autoimmune encephalomyelitis (EAE). Objectives: The objectives of the study were to determine molecular mechanisms underlying suppression of the encephalitogenic response by these drugs; likewise, whether clinically-relevant post-treatment paradigms with PARP-1 inhibitors could prevent EAE relapses. Methods: Adopted both in vitro techniques (bone marrow-derived cultured DC) as well as in vivo models of chronic or relapsing–remitting (RR) EAE. Results: We report that two structurally unrelated PARP-1 inhibitors negatively regulated NFκB activation, as well as maturation, cytokine production and APC function of cultured mouse bone marrow-derived dendritic cells (DCs). PARP-1 inhibitors also reduced the number and APC function of DCs migrating in the draining lymph nodes of ovalbumin-immunized mice. In C57Bl mice with chronic EAE or SJL mice with RR EAE, pharmacological inhibition of PARP-1 reduced CNS DC migration and demyelination as well as neurological impairment to an extent similar to that achieved with the potent immunosuppressant cyclosporine A. Remarkably, PARP-1 inhibitors injected after the first phase of disease reduced relapse incidence and severity, as well as the spinal cord number of autoreactive Th17 cells. Under this clinically-relevant treatment paradigm, PARP inhibitors also suppressed epitope spreading of the encephalitogenic response. Conclusions: Overall, data underscore the potential relevance of PARP-1 inhibitors to MS therapy and suppression of autoimmunity.

Time-course expression of metallothioneins and tissue metals in chronic relapsing form of experimental autoimmune encephalomyelitis.

To elucidate the role of metallothioneins (MTs) in the pathomechanisms of autoimmune CNS disorders we estimated the expression of MTs I+II and the tissue concentrations of Zn²+ and Cu²+ in the brain, spinal cord (SC) and in the liver during the periods of attacks and remissions in chronic relapsing experimental autoimmune encephalomyelitis (CR-EAE). Disease was induced in the genetically susceptible Dark Agouti (DA) rats by subcutaneous injection of bovine brain homogenate in CFA. Control rats were treated with CFA. The data, obtained by clinical assessment, immunohistochemistry and inductivity coupled plasma spectrometry, have shown that during the first attack (on the 12th day) MTs I+II were markedly upregulated in subarachnoid regions and perivascular space on astrocytes, microglia and on spinal neurons. Simultaneously, the concentrations of zinc in the SC and zinc and copper in the liver have found to be increased. During the second attack (on the 22nd day) a new overexpression of MTs was found in the cerebellum, in sulcus hippocampi, in spinal neurons and particularly in hepatocytes around the central vein. Concomitantly, in the brain and SC the concentration of copper increased. The data point to a neuroprotective role of MTs and to an important regulatory role of essential metals and hepatic MTs in the pathogenesis of CR-EAE.

Relapse of Experimental Autoimmune Encephalomyelitis after Discontinuation of FTY720 (Fingolimod) Treatment, but Not after Combination of FTY720 and Pathogenic Autoantigen

FTY720 (Fingolimod) is known to have a significant therapeutic effect on experimental autoimmune encephalomyelitis (EAE). Here, we used an EAE mouse model, which had been established by immunizing C57BL/6J mice with a partial peptide of myelin oligodendrocyte glycoprotein (MOG₃₅₋₅₅), to examine the relapse of EAE upon discontinuation of treatment with FTY720 alone or in combination with MOG₃₅₋₅₅. Relapse was confirmed to occur in all animals (n=6) within one week after discontinuation of FTY720, with increase in the number of lymphocytes infiltrating the spinal cord and demyelination. However, in the case of combination therapy with FTY720 and MOG₃₅₋₅₅, relapse following discontinuation of treatment was completely suppressed. The autoantigenic peptide might serve to suppress the clonal selection of relapse-associated autoantigen-specific T cells.

Acute and chronic cannabinoid extracts administration affects motor function in a CREAE model of multiple sclerosis

The multiple sclerosis is an immuno-mediated disorder of the Central Nervous System characterized by inflammatory processes and neurodegenerative changes. It has been shown that the endocannabinoid system is altered in this disease and that the exogenous cannabinoids may play a possible role in its therapeutic management. The aim of the present study was to investigate the efficacy of crude extracts of Cannabis sativa on motor symptoms in the chronic relapsing experimental autoimmune encephalomyelitis (CREAE), a murine model of multiple sclerosis. CREAE-induced mice were injected by different crude ethanolic extracts from Cannabis sativa, containing Δ⁹-tetrahydrocannabinol, cannabidiol, or cannabinoid-free, respectively. The effect of the combined treatment with Δ⁹-tetrahydrocannabinol and cannabidiol extracts has also been investigated. All extracts were administered in acute and chronic experimental protocols. The chronic administration of Δ⁹-tetrahydrocannabinol-rich extract resulted in a significant reduction of neurological deficits that lasted until the end of the observations. The acute and chronic treatments with the cannabidiol-rich extract resulted unable to induce changes of neurological signs. However, during the relapse phase a significant decrease of neurological scores was observed. The combined treatment with Δ⁹-tetrahydrocannabinol and cannabidiol extracts was ineffective, whereas the acute administration of the cannabinoid-free extract showed a significant improvement. Our study shows a patchy effect of different cannabinoid extracts on CREAE-induced motor deficits. Although the effect of crude extracts of Cannabis sativa here reported need to be further investigated to define the exact therapeutic target of each cannabinoid, it may represent a possible therapeutic approach for the management of the multiple sclerosis.

Fingolimod (FTY720), sphingosine 1-phosphate receptor modulator, shows superior efficacy as compared with interferon-β in mouse experimental autoimmune encephalomyelitis

Fingolimod (FTY720), a sphingosine 1-phosphate (S1P) receptor modulator, inhibits S1P-dependent lymphocyte egress from secondary lymphoid organs and is highly effective in experimental autoimmune encephalomyelitis (EAE) in mice. In this study, we directly compared the therapeutic effects of FTY720 and recombinant mouse interferon (rm-IFN)-β on relapse and progression of EAE in mice. When FTY720 at oral dose of 0.03 to 1 mg/kg was administered daily after establishment of EAE induced by myelin proteolipid protein (PLP) in SJL/J mice, relapse of EAE was significantly inhibited during administration period. Subcutaneous injection of rm-IFN-β (10,000 IU/mouse) also inhibited the relapse of EAE at early period; however EAE was relapsed in all the mice within administration period. Therapeutic administration of FTY720 (0.03 to 1 mg/kg) significantly improved the symptoms of chronic EAE induced by myelin oligodendrocyte glycoprotein in C57BL/6 mice whereas rm-IFN-β (10,000 IU/mouse) showed no clear effect. These results indicate that FTY720 is more efficacious in mouse EAE as compared with rm-IFN-β. FTY720 markedly reduced the frequency of PLP-specific Th17 and Th1 cells in the spinal cord of EAE mice. On the contrary, FTY720 increased the frequency of PLP-specific Th17 and Th1 cells in the inguinal lymph nodes, suggesting inhibition of egress of myelin antigen-specific Th cells from draining lymph nodes. From these results, the ameliorating effects of FTY720 on EAE are likely due to reduction of infiltration of myelin antigen-specific Th17 and Th1 cells into the central nervous system.

A Paradoxical Effect of Systemic IL-23 in EAE -; Limitation of Autoimmune Inflammatory Demyelination~!2009-11-30~!2010-02-16~!2010-07-14~!

The heterodimeric cytokine IL-23 plays a non-redundant function in the development of cell-mediated, organ- specific autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). To further characterize the mechanisms of action of IL-23 in autoimmune inflammation, we administered IL-23 systemically at different time points during both relapsing and chronic EAE. Surprisingly, we found suppression of disease in all treatment protocols. We ob- served a reduction in the number of activated macrophages and microglia in the CNS, while T cell infiltration was not significantly affected. Disease suppression correlated with reduced expansion of myelin-reactive T cells, loss of T-bet ex- pression, loss of lymphoid structures, and increased production of IL-6 and IL-4. Here we describe an unexpected func- tion of exogenous IL-23 in limiting the scope and extent of organ-specific autoimmunity.

Fingolimod (FTY720) ameliorates experimental autoimmune encephalomyelitis (EAE): I. Oral administration of FTY720 effectively inhibits relapse of EAE

Experimental autoimmune encephalomyelitis (EAE) is a CD4 T cell-mediated disease model for multiple sclerosis (MS). When SJL/J mice are immunized with myelin proteolipid protein (PLP), EAE symptoms were developed within 2 weeks, remitted thereafter, and then relapsed at about 3 weeks after immunization, indicating that EAE induced by PLP in SJL/J mice shares a certain characteristic with relapsing remitting MS. In this study, we evaluated the preventing effect of fingolimod (FTY720), a sphingosine 1-phosphate receptor modulator, on relapse of EAE induced by PLP in SJL/J mice. When FTY720 at oral does of 0.1 and 0.3 mg/kg was administered daily after establishment of EAE, relapse of EAE was markedly inhibited during administration period. The relapse of EAE was significantly inhibited by subcutaneous administration of recombinant mouse interferon-β (rm-IFN-β) at 10000 IU every other day at early period; however EAE was relapsed in the half number of mice in latter period. These results indicate that FTY720 shows a more marked preventing effect on relapse of EAE compared with rm-IFN-β. By immunohistochemical staining, it is revealed that the area of demyelination and the infiltration of CD4 T cells are significantly reduced in the spinal cords of EAE mice by FTY720. Interestingly, FTY720 markedly decreased infiltration of PLP-specific, interleukin 17-expressing CD4 T cells (Th17 cells) into the spinal cords. Consequently, the preventing effect of FTY720 on relapse of EAE is likely due to reduction of infiltration of encephalitogenic CD4 T cells into the central nervous system.

Mycobacterium smegmatisExpressing a Chimeric Protein MPT64-Proteolipid Protein (PLP) 139–151 Reorganizes the PLP-Specific T Cell Repertoire Favoring a CD8-Mediated Response and Induces a Relapsing Experimental Autoimmune Encephalomyelitis

We infected SJL mice with a recombinant Mycobacterium smegmatis expressing a chimeric protein containing the self-epitope of proteolipid protein 139-151 (p139) fused to MPT64, a secreted protein of Mycobacterium tuberculosis (rMS(p139)). Infected mice developed a relapsing experimental autoimmune encephalomyelitis (EAE), showing a prevailing demyelination of the CNS, and disease severity was significantly lower in comparison with the one that follows immunization with p139. rMS(p139) was not detected in lymph node or spleen in the course of clinical disease development or in the CNS during relapse. Infection with rMS(p139) modified the p139-specific T cell repertoire, recruiting the spontaneous p139-specific repertoire and activating CD4(+) T cells carrying the BV4 semiprivate rearrangement. T cells carrying the public BV10 rearrangement that are consistently found in the CNS during flares of disease were not activated by infection with rMS(p139) because lymph node APCs infected with rMS(p139) selectively fail to present the epitope for which BV10 cells are specific. Simultaneously, rMS(p139) expanded p139-specific CD8(+) cells more efficiently than immunization with peptide in adjuvant. SJL mice vaccinated against the CDR3 sequence of the BV10 public rearrangement reduced usage of the BV10 cells and displayed reduced symptoms during bouts of EAE. Thus, transient peripheral infection with a CNS-cross-reactive nonpathogenic Mycobacterium induces a relapsing EAE that continues long after clearance of the infectious agent. The composition of the self-reactive repertoire activated determines severity and histology of the resulting disease.