Course Of Experimental Allergic Encephalomyelitis Research Articles

The role of vitamin D through SphK1/S1P in the regulation of MS progression

Sphingosine-1-phosphate (S1P) is biologically active lipid, leading to neuroinflammation and macrophage invasion in central nervous system, plays an important role in the development of multiple sclerosis (MS) model in experimental allergic encephalomyelitis (EAE) rats. Vitamin D is observed to be a key factor in regulating cell S1P levels. We detected vitamin D can alleviate the symptoms of EAE rats, but the exact mechanism is unclear. In PC12 cells, vitamin D can reverse S1P-induced cell death, but the signaling pathway unclear. This study was aimed to investigate S1P regulation mechanism or signaling pathway mediated by vitamin D in EAE and PC12 model. In our experiments, S1P and Sphingosine kinase type 1 (SphK1) mRNA and protein expression in EAE rats group, control group, vitamin D feeding group were detected by HPLC, ELISA, RT-PCR and western blot. PC12 cell death was detected by Propidium (PI) staining. VDR plasmid overexpression and RNA interference, immunofluorescence, real-time cell analysis, protein immunoblotting was used to detect SphK1 transcriptional regulation, cell-substrate attachment quality, the signaling pathway of cell apoptosis and inflammation related gene expression (Bax/Bcl-2, Casepase-3, Il-6, TGF-β, TNF-α). Our study showed vitamin D can reverse the elevation of S1P level in EAE rats, reduce the severity and shorten the course of EAE. 1,25-(OH) 2D3 coupled with vitamin D receptor (VDR) inhibited SphK1 transcription. 1,25-(OH)2D3 significantly reduced PC12 cell death rate induced by S1P, in addition improved the cell substrate attachment quality. 1,25-(OH) 2D3 can block S1P-induced p-ERK activation and PI3K /Akt signaling pathway reduced Il-6, TGF-β, TNF-α cytokine release and Bax/Bcl-2, Casepase-3 apoptosis protein expression. On the other hand, immunofluorescence staining showed 1,25-(OH) 2D3 can increase the expression of neuronal perinuclear protein MAP2 in PC12 cells probably protect nerve cells further. In summary, the ameliorative effect of vitamin D was derived from its ability to reduce S1P levels, provides an idea for vitamin D as a combination therapy for disease.

Developmental ablation of mature oligodendrocytes exacerbates adult CNS demyelination

Multiple sclerosis (MS) is a CNS neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and the spinal cord that results in localized functional deficits. Several risk factors have been associated with MS, however none fully explain the enhanced susceptibility seen in older individuals. Epidemiological data, based on geographical prevalence studies suggest that susceptibility is established early in life and frequently long before the diagnosis of disease raising the possibility that developmental events influence adult disease onset and progression. Here we test the hypothesis that selective loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to a demyelinating insult to the mature CNS. A transgenic mouse model was utilized to specifically induce apoptotic cell death in a subset of mature oligodendrocytes (MBP-iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult animal. Immunostaining, immunoblotting, behavioral testing, and electron microscopy were utilized to examine the differences in the response between animals with developmental loss of oligodendrocytes and controls. We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10–14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte loss showed delayed recovery from an LPC lesion to the mature spinal cord. Unexpectedly, the induction and severity of MOG induced EAE was not significantly altered in animals following oligodendrocyte developmental loss even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is unclear why the elevated glial responses seen in developmentally compromised animals were not reflected in enhanced functional deficits. These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its response to subsequent insults and the capacity for repair in the adult.

Sarm1 knockout protects against early but not late axonal degeneration in experimental allergic encephalomyelitis.

Programmed axonal degeneration, also known as Wallerian degeneration, occurs in immune-mediated central nervous system (CNS) inflammatory disorders such as multiple sclerosis and the animal model experimental allergic encephalomyelitis (EAE). Sterile alpha and TIR domain containing protein 1 (SARM1) functions to promote programmed axonal degeneration. To test the hypothesis that loss of SARM1 will reduce axonal degeneration in immune-mediated CNS inflammatory disorders, the course and pathology of EAE was compared in Sarm1 knockout mice and wild type littermates. The clinical course of EAE was similar in Sarm1 knockout and wild type. Analysis of EAE in mice expressing neuronal yellow fluorescent protein (YFP) showed significantly less axonal degeneration in Sarm1 knockout mice compared to wild type littermates at 14 days post-induction of EAE. At 21 days post-induction, however, difference in axonal degeneration was not significant. At 42 days post-induction, Sarm1 knockout mice were indistinguishable from wild type with respect to markers of axonal injury, and were similar with respect to axonal density in the lumbar cords. There was no significant change in peripheral immune activation or CNS inflammatory cell infiltration associated with EAE in Sarm1 knockout mice. In conclusion, Sarm1 deletion delayed axonal degeneration early in the course of CNS inflammation, but did not confer long-term protection from axonal degeneration in an animal model of immune-mediated CNS inflammation.

The contribution of cyclophilin A to immune-mediated central nervous system inflammation

Cyclophilin A has multiple known functions in inflammation. Intracellular cyclophilin A modulates T helper 2 response (Th2) and extracellular cyclophilin A functions as a leukocyte chemotactic factor. The contribution of cyclophilin A to central nervous system (CNS) inflammation has not been reported. To test the hypothesis that inhibition of cyclophilin A would ameliorate immune-mediated CNS inflammation, we compared the course and neuroimmunology of experimental allergic encephalomyelitis (EAE) in cyclophilin A knockout mice and wild type littermates. There was a trend towards lower incidence of EAE in cyclophilin A knockout mice, but the clinical course of EAE among animals that manifested clinical signs of EAE was similar in cyclophilin A knockout and wild type littermates. Antigen recall response to myelin oligodendrocyte glycoprotein (MOG) peptide showed that interferon-γ release was lower in cyclophilin A knockout mice. Analysis of CNS inflammatory cells showed that CD3+ T cell infiltration into the CNS was lower in cyclophilin A knockout mice. These results showed that the loss of cyclophilin A results in altered peripheral immune activation and CNS leukocyte infiltration, but these changes did not result in a substantial change in the clinical course of EAE.

A Perspective of Coagulation Dysfunction in Multiple Sclerosis and in Experimental Allergic Encephalomyelitis.

A key role of both coagulation and vascular thrombosis has been reported since the first descriptions of multiple sclerosis (MS). Subsequently, the observation of a close concordance between perivascular fibrin(ogen) deposition and the occurrence of clinical signs in experimental allergic encephalomyelitis (EAE), an animal model of MS, led to numerous investigations focused on the role of thrombin and fibrin(ogen). Indeed, the activation of microglia, resident innate immune cells, occurs early after fibrinogen leakage in the pre-demyelinating lesion stage of EAE and MS. Thrombin has both neuroprotective and pro-apoptotic effects according to its concentration. After exposure to high concentrations of thrombin, astrocytes become reactive and lose their neuroprotective and supportive functions, microglia proliferate, and produce reactive oxygen species, IL-1β, and TNFα. Heparin inhibits the thrombin generation and suppresses EAE. Platelets play an important role too. Indeed, in the acute phase of the disease, they begin the inflammatory response in the central nervous system by producing of IL-1alpha and triggering and amplifying the immune response. Their depletion, on the contrary, ameliorates the course of EAE. Finally, it has been proven that the use of several anticoagulant agents can successfully improve EAE. Altogether, these studies highlight the role of the coagulation pathway in the pathophysiology of MS and suggest possible therapeutic targets that may complement existing treatments.

Efficiency of lithium salt of disulphide of glutathione and inosine in treatment of multiple sclerosis in experiment

We studied the possibility of pharmacological correction of neurological disorders in guinea pigs and rabbits with the lithium salt of disulphide of glutathione and inosine medication in experimental allergic encephalomyelitis caused by inoculation of the main myelin protein in Freund’s complete adjuvant. The intradermal test with the main protein of myelin was accomplished in all animals to confirm the autoimmune process at 21st day after sensibilization. The lithium salt of disulphide of glutathione and inosine medication was administered intramuscularly every other day at a single dose of 10 mg/kg for 5 weeks. The symptoms of experimental allergic encephalomyelitis were more pronounced in rabbits and to a lesser extent in guinea pigs. The high therapeutic efficacy of lithium salt of disulphide of glutathione and inosine was established according to the severity and incidence of neurological disorders (dysfunction of the pelvic organs, ataxia, tremor, paralysis and paresis of the limbs) and lethality rate. Early (at 5 days after sensibilization) and subsequent therapeutic use of lithium salt of disulphide of glutathione and inosine prevented the development of experimental allergic encephalomyelitis in 75% of laboratory animals, in the rest 25% of the animals the course of experimental allergic encephalomyelitis was comparatively mild. There were no lethal outcomes in study group compared with lethal outcomes in 70% of animals in control group. Experimental evidence of neuroprotective effects of lithium in brain ischemia and in neurodegenerative diseases highlights new opportunities for the use of lithium-containing drugs in clinical neurology. It has been confirmed that the lithium salt of glutathione and inosine disulfide has a moderate immunosuppressive effect, inhibiting the biosynthesis of antibodies and the delayed-type hypersensitivity reaction.

Cyclophilin inhibitor NIM811 ameliorates experimental allergic encephalomyelitis

Cyclophilins have diverse functions that may affect the course of central nervous system (CNS) inflammatory disorders. Anti-inflammatory and neuroprotective mechanisms may be targeted by inhibition of cyclophilin A-dependent and cyclophilin D-dependent functions, respectively. We tested the effect of cyclophilin inhibition on CNS inflammation by administering N-methyl-4-isoleucine-cyclosporin (NIM811) to mice undergoing experimental allergic encephalomyelitis (EAE). Treatment with NIM811 resulted in significant reduction of EAE clinical severity. Analysis of mitochondrial calcium retention capacity and the course of EAE in cyclophilin D knockout mice indicated that the effect of NIM811 on EAE was not entirely cyclophilin D-dependent. NIM811-treated EAE animals showed reduction in interleukin-2 expression and reduction in CNS inflammatory infiltrates. These results indicate that anti-inflammatory rather than neuroprotective mechanisms associated with cyclophilins are likely involved in the mechanism of NIM811 in EAE.

The features of experimental allergic encephalomyelitis after stem cells transplantation

The objective. To study the course of experimental allergic encephalomyelitis (EAE) after intravenous administration of hematopoietic stem cells (HSC) and suboccipital transplantation of fetal neurocells (FNC), and treatment impact on intensity of neural tissue renewing in rats with EAE. Materials and methods. The experiment included 40 rats, average weight 200 g. EAE was induced using complete Freund’s adjuvant. In 32 rats with EAE we used intravenous HSK (10 million cells or 20 million cells) and suboccipital transplantation of FNC (0.1 ml). The EAE clinical course was estimated up to 110 days. The rats were taken from the experiment on 23 rd , 25 th days and in 2 months after EAE induction. The preparations were stained by Nisse, with hematoxylin, eosin and picro-fuchsin. Morphological and morphometric studies were performed. Results. Complete recovery was not observed, and on the 26th day of the experiment EAE was regarded as a chronic process. After allogeneic HSC administration (in dose 10 million cells) peak of clinical manifestations was observed on the 20 th day of the experiment with animals’ gradual complete recovery. Administration of allogeneic HSC in a dose of 20 million cells leads to significantly more rapid regression of clinical signs compared to the treatment with allogeneic HSC in a dose of 10 million cells. Animals’ recovery after intravenous introduction of HSC and suboccipital transplantation of FNC was significantly more rapid than after treatment just with HSC. The treatment with intravenous introduction of HSC (20 million cells) and suboccipital transplantation of FNC was associated with temporary significant deterioration after HSC administration and rapid recovery after FNC application (on the 29 th day). In all treatment options we revealed the tendency to reduction of pathologically changed neurocyts and gliocyts number. Conclusion. Suboccipital administration of allogeneic FNC improves the clinical condition of rats with EAE. The best results of experimental EAE treatment were observed at intravenous administration of HSC (20 million cells) followed by FNC neurotransplantation, particularly on the 63 rd day.

Potential Role of Nitrobiolome in Autoimmune Disease: Efficacy of GSNO in EAE.

Abstract S-nitrosoglutathione (GSNO) is a physiological nitric oxide (NO) carrier molecule important in modulation of NO-signaling. Cellular GSNO homeostasis is maintained by its synthesis by nitric oxide synthase (NOS) as well as its catabolism by GSNO reductase (GSNOR). Treatment of EAE mice (wild-type /WT) with exogenous GSNO or GSNOR inhibitor (N6022) significantly alleviated the clinical disease and associated myelin damage. In addition, more profound alleviation of EAE disease was observed in mice deficient in GSNOR (GSNOR−/−). However, EAE disease was further exacerbated in mice deficient in inducible NOS (iNOS−/−) as compared to WT mice. As expected, the spleen levels of S-nitrosothiol proteins were higher in GSNOR−/− EAE mice but lower in iNOS−/− EAE mice compared to WT EAE mice indicating a negative correlation of spleen GSNO levels with EAE disease severities. Both GSNO and N6022 treated EAE mice had decreased TH17 and increased CD4+/CD25+/FOXP3− Treg differentiations without any significant alterations in other CD4+ T cell subset differentiation. Accordingly, CD4 T+ cells from GSNO or N6022 treated EAE mice produced lower amount of IL-17 and higher amount of IL-10 compared to the cells from untreated WT EAE mice. GSNOR−/− EAE mice also exhibited decreased T 17 and increased CD4+/CD25+/FOXP3− Treg differentiations. On the other hand, iNOS−/− EAE mice exhibited increased TH17, but decreased Tregs differentiations compared to WT EAE mice. In summary, these studies document that nitrobiolome (GSNO) participates in differentiation of autoreactive TH17 and Treg subsets during the course of EAE, and that GSNO as a potential therapeutic drug for multiple sclerosis. Supported in-part by VA(BX002829) and NIH(NS037766).

Release-Active Antibodies to S100 Protein Can Correct the Course of Experimental Allergic Encephalomyelitis

Objective. To study the efficacy of release-active forms of antibody to S100 protein in an animal model of multiple sclerosis. Materials and methods. The study was performed on 60 female rats aged 12 weeks. Experimental allergic encephalomyelitis was induced by administration of spinal cord homogenate at the base of the tail. Female rats then received intragastric solution of release-active antibodies to S100 protein (Tenoten) at a dose of 2.5 ml/kg/day or distilled water for 30 days. Positive controls received i.m. injections of glatiramer acetate at a dose of 4 mg/kg/day (Copaxone). Results and discussion. Administration of release-active forms of antibody to S100 increased the latent period of disease onset, decreased in peak disease intensity, and compensated for weight loss in the animals, as compared with controls. The effect of the agents was comparable with that of the Copaxone group.

The Course of Experimental Allergic Encephalomyelitis in Adult Rats after Administration of Interleukin-1β at Different Periods in Early Life

Multiple sclerosis (MS) is a chronic disease of the CNS affecting people of working age, in which myelin and the cells producing it, and also neurons, become the targets for aggressive immune cells. It has been suggested that common childhood infections in later childhood increase the risk of developing MS. We report here studies of the course of experimental allergic encephalomyelitis (EAE) in rats given interleukin-1β (IL-1β) at different stages of early postnatal ontogeny. EAE was induced in rats at age three months by single subcutaneous immunizations with homologous spinal cord homogenate in complete Freund’s adjuvant. The number of sick animals was recorded daily, as were the severity and duration of disease. EAE was found to have a more severe course after administration of IL-1β in weeks 1 and 4 of life than in rats of the corresponding control groups. The harmful or protective consequences of IL-1β administration at different periods of early postnatal ontogeny are discussed, as are the role of stress reactivity and its link with the “hygiene hypothesis.”

Alpha-methylacyl-CoA racemase deletion has mutually counteracting effects on T-cell responses, associated with unchanged course of EAE.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system. Altering the metabolism of immune cells is an attractive strategy to modify their activity during autoimmunity in MS. We investigated the effect of modulating fatty acid metabolism in an animal model of MS, EAE. Alpha-methylacyl-CoA racemase (AMACR) converts R-configuration branched fatty acids into the S-configuration, thereby preparing them for β-oxidation. We observed a significant, disease-dependent elevation of AMACR expression in monocytes and T cells from blood, draining lymph nodes and spleen of EAE mice during the preclinical phase. In vitro analysis revealed that the proliferation of T cells was inhibited in AMACR KO mice, but T-cell polarization was switched toward a pathogenic state involving the production of more IFN-γ and IL-17, but less IL-4. These opposing effects appeared to cancel out each other in vivo, because AMACR KO EAE mice showed a marginal increase in the severity of early clinical symptoms. AMACR was not regulated in the white blood cells of MS patients. Our data show that AMACR is regulated in immune cells during EAE, but it is not a suitable target for the treatment of MS due to its opposing effects.

NFAT1 deficit and NFAT2 deficit attenuate EAE via different mechanisms.

EAE serves as an animal model for multiple sclerosis and is initiated by autoreactive T cells that infiltrate the CNS. Recognition of myelin-associated Ags within the CNS leads to activation of the transcription factor family NFAT. Here, we demonstrate an essential role for NFAT in disease induction, as the combined lack of NFAT1 (NFATc2) and NFAT2 (NFATc1) completely protected mice. Single deficiency of either NFAT1 or NFAT2 ameliorated the course of EAE, and NFAT2 ablation resulted in an obstructed proinflammatory reaction. However, NFAT1 deficit led to an anti-inflammatory response with nonpathogenic Th17 and Th2 cells concurrently secreting IL-17, IL-4, and IL-10. Both IL-4 and IL-10 contributed to disease protection. In Nfat1(-/-) CD4(+) T cells, the expression of anti-inflammatory lymphokines was mediated by NFAT2, thus directly enabling protective IL expression. Consequently, blocking NFAT in toto may be an option for immunosuppressive therapy. More importantly, selective NFAT1 blockade could represent a safe long-term immunomodulatory treatment approach for multiple sclerosis patients, potentially avoiding the adverse effects of global immunosuppression.

The experimental antiphospholipid syndrome: an invaluable tool to study autoimmunity-induced neurodegeneration

cells and its activation inhibits their differentiation and remyelination. These suggest a possible role of CNS TLR2 in progressive autoimmune demyelination. Methods: We examined the effects of intra-cerebro-ventricular (ICV) injection of Zymozan, a TLR2 agonist, on the clinical and pathological course of EAE. The survival and clinical scores were monitored; demyelination and axonal loss were quantified by gold-black and Bielschowsky stains, and the nature of neuro-inflammatory response was characterized by TLR2, IBA-1 and CD3 stainings and PCR for immune cytokines. Immune cells were isolated from EAE brain tissue and their proliferative response to the autoantigen (PLP peptide) or Concavallin A was examined in vitro. Results: Zymozan injection in naive mice induced a strong neuroinflammatory response without any clinical manifestations. In EAE mice, ICV Zymozan induced a severe acute toxic response with 80% mortality. Surviving animals returned to pre-injection clinical score, and their course of disease and CNS inflammatory parameters were not altered as compared to control EAE group. Demyelination and axonal loss were not affected by ICV Zymozan injections. Quantification of immune response in the brain by real time PCR, immunofluorescent stains and proliferative response to PLP peptide and ConA indicated an increase in innate but not adaptive immune response. Conclusions: (1) EAE mice are hypersensitive to CNS TLR2 activation with a severe toxic response. This might represent the susceptibility of multiple sclerosis patients to even trivial infections. (2) CNS TLR2 activation does not alter the clinical and pathological course of EAE. (3) These findings imply that CNS TLR2 activation affects the innate but not adaptive brain immune responses.

Protein kinase C θ regulates the phenotype of murine CD4+ Th17 cells.

Protein kinase C θ (PKCθ) is involved in signaling downstream of the T cell antigen receptor (TCR) and is important for shaping effector T cell functions and inflammatory disease development. Acquisition of Th1-like effector features by Th17 cells has been linked to increased pathogenic potential. However, the molecular mechanisms underlying Th17/Th1 phenotypic instability remain largely unknown. In the current study, we address the role of PKCθ in differentiation and function of Th17 cells by using genetic knock-out mice. Implementing in vitro (polarizing T cell cultures) and in vivo (experimental autoimmune encephalomyelitis model, EAE) techniques, we demonstrated that PKCθ-deficient CD4+ T cells show normal Th17 marker gene expression (interleukin 17A/F, RORγt), accompanied by enhanced production of the Th1-typical markers such as interferon gamma (IFN-γ) and transcription factor T-bet. Mechanistically, this phenotype was linked to aberrantly elevated Stat4 mRNA levels in PKCθ−/− CD4+ T cells during the priming phase of Th17 differentiation. In contrast, transcription of the Stat4 gene was suppressed in Th17-primed wild-type cells. This change in cellular effector phenotype was reflected in vivo by prolonged neurological impairment of PKCθ-deficient mice during the course of EAE. Taken together, our data provide genetic evidence that PKCθ is critical for stabilizing Th17 cell phenotype by selective suppression of the STAT4/IFN-γ/T-bet axis at the onset of differentiation.

Distinct roles of IFNβ and IFNγ in the production of proinflammatory and antiinflammatory cytokines in bone marrow-derived dendritic cells (BA12P.109)

Abstract IFNβ and IFNγ play distinct roles in MS/EAE. IFNγ exacerbates whereas IFNβ ameliorates the disease of EAE. Dendritic cells (DC) paly a sentinel role in the immune system, and are recruited to the CNS to restimulate encephalitogenic T cells during the course of EAE. We investigate how DC are regulated differently by IFNβ and IFNγ on the production of proinflammatory cytokines IL-12 and IL-23 and of antiinflammatory cytokine IL-10. Moreover, the molecular mechanisms of IFNβ and IFNγ-mediated regulatory effects are also investigated. Our results show IFNβ inhibited the production of IL-12 and IL-23, whereas enhanced the production of IL-10 in activated DC. Although both IFNβ and IFNγ inhibited IL-23 production, IFNγ enhanced IL-12 but suppressed IL-10 production. IFNγ-induced STAT-1/IRF-1 activation is required for IL-12 upregulation and IL-23 downregulation. Although STAT-1 activation is responsible for IFNγ-mediated IL-10 downregulation, IRF-1 activation is not involved in STAT-1-mediated IL-10 downregulation. Similar to IFNγ, IFNβ induced STAT-1 activation to suppress IL-23 production, however, the effect is independent of IRF-1 activation. IFNβ-induced Akt activation and GSK3β inactivation might be responsible for IL-12 downregulation and IL-10 upregulation as GSK3β activation is linked to inflammatory responses. In conclusion, our results provide the molecular mechanisms by which IFNβ and IFNγ utilize distinct signaling pathways to regulate the cytokine productions in DCs.

Mycophenolate mofetil improves neurological function and alters blood T-lymphocyte subsets in rats with experimental autoimmune encephalomyelitis

This study evaluated the clinical and pathological effects of the immunosuppressive agent mycophenolate mofetil (MMF) in rats with experimental autoimmune encephalomyelitis (EAE; a model of multiple sclerosis [MS]). EAE rats were randomly divided into 4 groups: model alone (n = 7); low- or high-dose MMF (20 and 30 mg/kg per day, respectively, n = 6 each) orally for 14 days; methylprednisolone (20 mg/kg per day, n = 6) injected once daily for 3 days. Six normal Wistar rats served as controls. Clinical signs and histopathological findings were evaluated 14 days after treatment started. Oral administration of high-dose MMF significantly ameliorated the course of EAE in rats: cumulative clinical scores were lower and weight loss was less than in rats receiving methylprednisolone. The ameliorated disease course was associated with alleviation of histopathological signs of EAE. Treatment increased the blood proportion of CD8(+), CD4(+)CD25(+) and CD4(+)CD45RA(+) T cells, with a concomitant reduced proportion of CD4(+) T cells and ratio of CD4(+) to CD8(+) T cells, compared with EAE model alone rats. MMF may have pharmacological potential in MS treatment and these findings may help in understanding the pathophysiological mechanism of MS.

Ethyl-eicosapentaenoic acid ameliorates the clinical course of experimental allergic encephalomyelitis induced in dark agouti rats

Eicosapentaenoic acid (EPA), a fatty acid present in high amount in fish, modulates immune response and stimulates myelin gene expression. In the present paper, we investigated the effects of EPA in an established animal model for multiple sclerosis (MS): experimental autoimmune encephalomyelitis (EAE) induced in dark agouti rats. Diets supplemented either with 0.2% or 0.4% of EPA were administrated daily from the day of induction until the end of experiment. One group of rats received diet supplemented with 0.2% of EPA 10 days before induction. The control group (immunized rats) was fed with chow diet. The animals were analyzed at two different stages of the disease: during the acute phase (14 d.p.i.) and during the recovery phase (32 d.p.i.). We showed a delayed onset of clinical severity of disease in all groups of rats fed EPA-supplemented diets. This effect was associated to an increased expression of myelin proteins and an improved integrity of the myelin sheath as well as an up-regulation of FoxP3 expression in the central nervous system during the acute phase of EAE. No significant changes in T cell subsets were noted at the periphery. On the contrary, during the recovery phase of EAE, in animals assuming EPA-supplemented diet, an increase of CD4+CD25+ and CD4+CD25+FoxP3+ in peripheral lymphocytes was noted. Our results indicate that EPA-supplemented diets may provide benefits to MS patients.

Analyzing the contributions of TCR affinity to the pathogenesis of experimental autoimmune encephalomyelitis (P4129)

Abstract Myelin oligodendrocyte glycoprotein (MOG) specific CD4+ T cell infiltrates from the central nervous system (CNS) have exhibited a range of affinities (low to high) for MOG peptide:MHC class II during peak EAE symptoms. Tetramer binding analyses and two-dimensional affinity assays have indicated that high affinity, MOG specific T cells comprise a rare yet significant population within the polyclonal repertoire; however, their functional contributions to the pathogenesis of chronic autoimmunity is not clear. Preliminary experiments suggested that a high precursor frequency (15 x 106) of polyclonal tetramer positive MOG specific T cells promotes early onset and greater disease severity when compared to tetramer negative T cells. Analysis of a high affinity MOG specific, CD4+ T cell (TCR Vα4β1) revealed a two dimensional affinity of 6.4x10-4 μm4 that was similar to the mean affinity (1.6x10-4 μm4) of pathogenic CD4+ T cells at peak LCMV Armstrong infection. Retrogenic models were used to assess the pathogenicity of TCR affinity in order to circumvent the limited number of endogenous high affinity T cells. The TCR Vα4β1 retrogenic mice developed spontaneous EAE even in the presence of FoxP3 positive (6%) T cells. T cells progressed though thymic selection without altering CD4 or TCR expression while maintaining their high two-dimensional affinity. Overall, MOG specific, high affinity CD4+ T cells are pathogenic and may predispose mice to a rapid progressive course of EAE.

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.