Experimental Allergic Encephalomyelitis Model Research Articles

Hyperforin down-regulates effector function of activated T lymphocytes and shows efficacy against Th1-triggered CNS inflammatory-demyelinating disease

Hyperforin (Hyp) is an active compound contained in the extract of Hypericum perforatum, well known for its antidepressant activity. However, Hyp has been found to possess several other biological properties, including inhibitory effects on tumor invasion, angiogenesis, and inflammation. In this paper, we show that treatment with Hyp inhibited IFN-gamma production, with down-regulation of T-box (T-bet; marker of Th1 gene expression) and up-regulation of GATA-3 (marker gene of Th2) on IL-2/PHA-activated T cells. In parallel, we showed a strong down-regulation of the chemokine receptor CXCR3 expression on activated T cells. The latter effect and the down-modulation of matrix metalloproteinase 9 expression may eventually lead to the inhibition of migratory capability and matrix traversal toward the chemoattractant CXCL10 by activated lymphocytes that we observed in vitro. The effect of Hyp was thus evaluated on an animal model of experimental allergic encephalomyelitis (EAE), a classic, Th1-mediated autoimmune disease of the CNS, and we observed that Hyp attenuates the severity of the disease symptoms significantly. Together, these properties qualify Hyp as a putative, therapeutic molecule for the treatment of autoimmune inflammatory disease sustained by Th1 cells, including EAE.

Effect of erhuangfang on cerebral and spinal demyelination and regeneration as well as expression of glial fibrillary acidic protein in rats with experimental allergic ncephalomyelitis

Background It demonstrates that erhuangfang can improve clinical symptoms of multiple sclerosis and relieve side effects of hormone. However, whether erhuangfang can improve experimental allergic encephalomyelitis (EAE) or not needs a further study. Objective To observe the effect of erhuangfang on neuro-pathology and astrocyte in EAE rats and compare with the effect of hormone. Design Randomized controlled animal study. Settings Department of Traditional Chinese Medicine, Beijing Tiantan Hospital, Capital Medical University; College of Traditional Chinese Medicine, Capital Medical University. Materials The experiment was carried out in the Laboratory Center of Capital Medical University from August to October 2005. Ten adult guinea pigs (SPF grade, weighing 400–450 g) and 70 adult Lewis rats (SPF grade, weighing 200–220 g) were selected in this study. Erhuangfang consisted of jiudahuang, shengdi, shuizhi, dabeimu, etc. Methods ▪ Experimental intervention: Rats were randomly divided into normal group ( n=10), model group (n=20), western medicine group ( n=20) and Chinese herb group ( n=20). Mixed emulsion, which was consisted of Freund's adjuvant and spinal cord homogenate of guinea pigs, was subcutaneously injected into palms of the two hindfeet of rats in the latter three groups to establish EAE models. Foot pads were injected with saline and then rats were perfused with saline in the normal group. In the model group, models were established as the same as those mentioned above, and rats were also perfused with saline. Rats in the western medicine group were perfused with saline and then 5 mg/kg prednisone acetate suspension. Rats in the Chinese herb group were perfused with erhuangfang decoction (15 g raw materials per kilogram) at 5 days before model establishment. The dosage in the four groups was 3 mL/day per rat. ▪ Experimental evaluation: At 28 days after model establishment, rats were randomly selected for cerebral (mainly surrounding cerebral ventricle) and spinal cord (cervical enlargement and lumbar enlargement) collections, and then haematine-eosin (HE) staining and SLG myelin staining were used to observe demyelination and regeneration; meanwhile, immunohistochemical staining was used to observe the expression of glial fibriliary acidic protein (GFAP). Main outcome measures Cerebral and spinal demyelination and regeneration as well as expression of GFAP in EAE rats. Results All 70 Lewis rats were involved in the final analysis. ▪ Demyelination and regeneration: Infiltration of inflammatory cells surrounding cerebrum and small venous vessels of spinal cord white matter, demyelination surrounding vessels and plentiful foam cells at myelinolysis sites were observed in the model group. Symptoms were relieved in the western medicine group and the Chinese herb group as compared with those in the model group. While, numbers of inflammatory infiltrated cells and vascular cuffs were decreased in focal region as compared with those in the model group; in addition, areas of softening focus and demyelination were decreased. ▪ Expression of GFAP: Volumes and numbers of positive cells of GFAP in white matter region were respectively bigger and higher than those of normal cells in the model group. Plentiful positive cells of GFAP were disorderly aggregated in hippocampus and surrounding small vessel cuffs. While, expression of GFAP was mildly increased surrounding focus in the Chinese herb group; however, GFAP did not express surrounding focus in the western medicine group. In addition, expressions of GFAP were not increased in non-focal region in both Chinese herb group and western medicine group. Conclusion Both erhuangfang and hormone can relieve inflammatory reaction of central nervous system and demyelination of EAE rats. On one hand, erhuangfang can regulate reaction of astrocyte in two ways, relieve reaction and proliferation of astrocyte in non-focal region and maintain the protective effect of astrocyte on brain tissue in focal region; on the other hand, hormone can overall inhibit reaction of astrocyte.

Regulated expression of the interferon-β gene in mice

A single plasmid regulated expression vector based upon a mifepristone-inducible two plasmid system, termed pBRES, has been constructed and tested in mice using murine interferon-b (mIFNb) as the transgene. The expression of mIFNb in the circulation was followed by measuring the systemic induction of IP-10, a validated biomarker for mIFNb in mice. Long-term, inducible expression of mIFNb was demonstrated following a single intramuscular (i.m.) injection of the pBRES mIFNb plasmid vector into the hind limb of mice. Induction of mIFNb expression was achieved by administration of the small molecule inducer, mifepristone (MFP). Plasmid DNA and mIFNb mRNA levels in the injected muscles correlated with mIFNb expression as monitored by IP-10 over a 3-month time period. Renewable transgene expression was achieved following repeat administration of the plasmid at 3 months following the first plasmid injection. A dose-dependent increase in expression was demonstrated by varying the amount of injected plasmid or the amount of the inducer administered to the mice. Finally, the pBRES plasmid expressing mIFNb under control of the inducer, MFP, was shown to be efficacious in a murine model of experimental allergic encephalomyelitis, supporting the feasibility of gene-based therapeutic approaches for treating diseases such as multiple sclerosis.

The essential role of mast cells in orchestrating inflammation

The essential role of mast cells in orchestrating inflammation

Minocycline Down-regulates MHC II Expression in Microglia and Macrophages through Inhibition of IRF-1 and Protein Kinase C (PKC)α/βII

Minocycline Down-regulates MHC II Expression in Microglia and Macrophages through Inhibition of IRF-1 and Protein Kinase C (PKC)α/βII

Connexin 43 gap junction proteins are up‐regulated in remyelinating spinal cord

Alterations in the expression of gap junction proteins have previously been observed in several diseases affecting the central nervous system; however, the status of connexin 43 (Cx43) has not yet been reported in spinal cord remyelination. We studied Cx43 expression in demyelination and remyelination by using a chronic guinea pig model of experimental allergic encephalomyelitis (EAE). Hartley guinea pigs were immunized with homogenized whole CNS and complete Freund's adjuvant. Animals became chronically ill by day 40 postimmunization, and animals with paralysis were entered into the study. Animals were treated on days 40-60 postimmunization with either saline or drugs that promote remyelination: an adenosine amine congener (100 mug/kg), an anti-alpha4-integrin blocker (CT301; ELN 69299; 30 mg/kg), or a combination of both drugs. Remyelination was induced in all drug-treated groups. Cx43 expression was virtually absent in demyelinated lesions of saline-treated controls compared with healthy tissue and normal appearing white matter (P < 0.001), whereas Cx43 was considerably increased (300-500%) in remyelinating lesions of all treatment groups (P < 0.001), most notably in CT301-treated animals. These changes in Cx43 expression indicate that Cx43 may beimportant for recovery from neuroinflammation.

Gene-Based Delivery of IFN-β Is Efficacious in a Murine Model of Experimental Allergic Encephalomyelitis

Experimental allergic encephalomyelitis (EAE) is a model of central nervous system (CNS) inflammation that follows immunization with certain CNS antigens. The course and clinical manifestations of EAE are similar to those of multiple sclerosis (MS) in humans; therefore, EAE has become an accepted animal model to study MS. The purpose of this study was to demonstrate that systemic expression of murine interferon-beta (IFN-beta) (MuIFN-beta), following intramuscular (i.m.) delivery of plasmid DNA encoding MuIFN-beta to the hind limb of mice, is effective in reducing the clinical manifestations of disease in a model of EAE. The results of the study demonstrate that gene-based delivery of MuIFN-beta caused significantly decreased clinical scores compared with delivery of the null vector. A single injection of the MuIFN-beta plasmid was as effective in reducing the severity of the disease as an every other day injection of MuIFN-beta protein.

A Tumor Necrosis Factor Receptor 1-Dependent Conversation between Central Nervous System-Specific T Cells and the Central Nervous System Is Required for Inflammatory Infiltration of the Spinal Cord

We examined the role of tumor necrosis factor receptor 1 (TNFR1) in inflammation initiated by the adoptive transfer of central nervous system (CNS)-specific Th1 cells in experimental autoimmune encephalomyelitis, a murine model of multiple sclerosis. This adoptive transfer paradigm eliminates the confounding effects of bacterial adjuvants in the analysis of inflammation. We found that although T cells could reach the meninges and perivascular space in the absence of TNFR1, recruitment of other inflammatory cells from the blood was dramatically reduced. The reduction in the recruitment of CD11b(hi) cells correlated with a dramatic reduction in the production of the chemokines CCL2 (MCP-1) and CXLC2 (MIP-2) in TNFR1-deficient hosts. Bone marrow chimera experiments demonstrated that TNF can be effectively supplied by either the hematopoietic system or the CNS, but the essential TNFR1-responsive cells reside in the CNS. Previous work has demonstrated that microglia produce CCL2, and here we demonstrate that astrocytes and endothelial cells produced CXCL2 in the early stages of inflammation. Therefore, productive inflammation results from a conversation, or mutually responding signals, between the initiating T cells and cells in the parenchyma of the spinal cord.

Intrasplenic electro-transfer of IL-4 encoding plasmid DNA efficiently inhibits rat experimental allergic encephalomyelitis

Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited only marginal therapeutic effects, if any, in the EAE model. One strategy to overcome this limitation would be to determine the optimal delivery route leading to significant beneficial effects both in early (prophylactic) and late (therapeutic) treatments. To address this issue, we directly compared the effects of intrasplenic (IS) and intramuscular (IM) electro-transfer of interleukin-4 (IL-4) DNA in the rat experimental allergic encephalomyelitis (EAE) model. In the preventive experiment, rats received IM (25 or 150 μg) or IS (25 μg) administration of IL-4 DNA followed by in vivo electroporation the day before MBP immunization. In the late treatment experiment, rats were treated with IM (150 μg) or IS (25 μg) administration of IL-4 DNA with electroporation 10 days after MBP immunization. As a control the same amount of vector DNA was used. Macroscopic analysis indicated that the onset of moderate to severe EAE in rats treated with IS transfer of 25 μg of IL-4 DNA was prevented on a significant level compared with IM 25 μg of the IL-4 DNA transfer group or the control group in the preventive experiments. More importantly, IS transfer of 25 μg of IL-4 DNA considerably suppressed the severity of EAE in late treatment experiments while IM transfer of 150 μg of IL-4 DNA had little effect. The MBP-specific expression of IFN-γ from stimulated splenocytes was considerably decreased by the IS IL-4 DNA transfer group both in the preventive and therapeutic experiments while IM transfer had this effect only in the preventive protocol. Histological analysis showed that spinal cord inflammation was considerably reduced in the IS IL-4 DNA transfer group. These data provide the first demonstration that IS electro-transfer of IL-4 DNA is more effective both in the prevention and modulation of EAE than IM transfer and that IS electro-gene transfer may present a new approach to cytokine therapy in autoimmune diseases.

Restrictive and diversifying elements of the anti-myelin/oligodendrocyte glycoprotein antibody response in primate experimental allergic encephalomyelitis

Autoantibody responses against conformational epitopes of myelin/oligodendrocyte glycoprotein (MOG) possess myelin destructive potential, as demonstrated in the marmoset model of human multiple sclerosis (MS) and in some rodent models of experimental allergic encephalomyelitis. We have previously characterized monoclonal Fab fragments specific for conformational epitopes of MOG that were derived from a combinatorial antibody library generated from a MOG-immune marmoset. In this paper, we address the molecular heterogeneity of humoral responses against MOG in this outbred model of MS by studying additional antibody clones derived from a genetically unrelated animal. We find that all MOG-specific IgGkappa Fab fragments, unrelated to genetic make-up, utilize a restricted set of variable region genes, IGHV1 and IGHV3 for the H chain and IGKV1, IGKV3, and IGKV5 for the L chain. Despite these restricting factors, diversity within these antibody repertoires can be observed, predominantly within the H-chain CDR3 regions. Our findings suggest that only a limited set of Ig genes is necessary to launch a diverse, destructive humoral immune response against a single CNS antigen in primates. These results are the first to contribute to a better understanding of how myelin-directed and potentially destructive autoantibody responses may develop in human MS.

Serum uric acid and multiple sclerosis

Peroxynitrite (PN) has been implicated in multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis. Uric acid (UA) serum levels of MS patients, a natural scavenger of PN, were found lowered in some recent studies. Objective/purpose The objective of our study was to correlate UA serum levels and several clinical parameters of MS. We also tried to investigate serum UA changes during treatment with immunomodulating or immunosuppressing drugs in the last 6 months. Patients and methods We measured UA serum levels in 190 patients with MS and 58 age and gender matched patients with inflammatory (IND) and non-inflammatory diseases (NIND) studied as control groups. UA levels were correlated with clinical parameters as type of the disease, duration, disability, magnetic resonance imaging (MRI) activity and female gender. Results In the overall MS group, patients were found to have significantly lower mean serum uric acid levels compared with the IND ( p = 0.0029) and the NIND group ( p < 0.0001). UA serum concentrations were not inversely correlated with duration of the disease ( p = 0.87), with disability as assessed by Expanded Disability Status Scale (EDSS) score ( p = 0.67) and MRI activity ( p = 0.36). Treatment with immunomodulating or immunosuppressing drugs had no influence in UA levels ( p = 0.85). Patients with Clinically Isolated Syndromes (CIS) were found to have significantly lower UA concentrations compared with IND and NIND patients ( p = 0.009 and <0.001, respectively). Conclusions Our findings suggest that lower serum UA levels in MS patients may represent a primary, constitutive loss of protection against nitric oxide and the development of CNS inflammation and tissue damage may not have a direct effect to UA serum levels. They also provide support that the earlier increase of UA serum levels might be beneficial in the future treatment of MS.

Improved detectability of experimental allergic encephalomyelitis in excised swine spinal cords by high b-value q-space DWI

Experimental allergic encephalomyelitis (EAE) is the primary experimental model of multiple sclerosis (MS), which involves both inflammation and demyelination and is known to be species-dependent. Spinal cord abnormalities were found in more than 80% of postmortem specimens of MS patients. In the present study, T 1, T 2 and high b-value q-space diffusion-weighted magnetic resonance imaging (MRI) were used, for the first time, to characterize the EAE model in excised swine spinal cords. The MR images were compared with histological staining and clinical scoring. Although all spinal cords were excised from swine with severe or very severe (clinical score between 3 to 5 on a scale of 5) motor impairments, T 1- and T 2-weighted MRI revealed white matter (WM) abnormalities in only five of the ten EAE diseased spinal cords studied, while high b-value q-space diffusion weighted MRI ( q-space DWI) detected WM abnormalities in all diseased spinal cords studied. Interestingly, high b-value q-space DWI was able to detect abnormalities in the normal appearing white matter (NAWM) even in spinal cords where no plaques were identified by the T 1- and T 2-weighted MR images. Good anatomical correlation was observed between the high b-value q-space MR images and histology. The extent of DWI abnormalities paralleled the clinical scoring and correlated with histology. In addition, areas classified as NAWM by the T 1- and T 2-weighted MR images that showed abnormalities in the q-space DWI were also found to have abnormal histology. This improved detection level of the EAE model by high b-value q-space DWI over conventional T 1-, and T 2-weighted MRI is briefly discussed.

Multiple sclerosis: looking beyond autoimmunity

The chronic incurable disorder multiple sclerosis (MS) is characterized by neurodegeneration, multifocal demyelination and astroglial proliferation (gliosis).2 The prevalence of MS is influenced by geography and genetics. In the Western world it is a leading cause of neurological disability in the young. The cause and exact pathogenesis are still unknown. Some see MS as a T-cell-driven autoimmune inflammatory disease, targeting the myelin sheaths in the central nervous system,3 but there is no proof. Unlike autoimmune conditions such as rheumatoid arthritis, systemic lupus erythematosus or myasthenia gravis, MS has no specific immunological marker.2,4 An animal model that has been used in MS research is experimental allergic encephalomyelitis (EAE), in which demyelination is induced by sensitization against myelin basic protein. Clinically and pathologically, however, EAE resembles acute disseminated encephalomyelitis (ADEM) rather than MS.2 Nonetheless, the EAE model has been used to drive the autoimmune theory and to develop treatments. An inflammatory hypothesis of demyelination also fails to explain various salient features of the disease (Box 1).5–7 Here, we explain our view that research and treatment strategy in MS need to change direction.

Animal Models of Central Nervous System Immune-Mediated Diseases: Therapeutic Interventions with Bioactive Peptides and Mimetics

Experimental allergic encephalomyelitis (EAE) is a T helper 1 (Th1) mediated autoimmune disease and the principal animal model for multiple sclerosis (MS). Like MS, EAE is characterized by a coordinated inflammatory attack on the myelin sheath in the central nervous system (CNS), with damage to axons. No matter whether the ideal animal model is not yet available, much knowledge concerning the pathogenesis of MS has been achieved through studies on EAE. Dissecting the underlying immune mechanisms provided recognition of several myelin antigens that are vulnerable in autoimmune attack. The beneficial effect and the mechanism of action of a number of the currently used immunomodulating agents in MS therapy were first indicated in EAE. Altered peptide ligands (APL) can modulate T-cell responses to native peptide antigens implicated in the pathogenesis of autoimmune diseases such as MS and EAE. However, peptide therapy is hindered due to the sensitivity of peptides to proteolytic enzymes as well as due to some immune-mediated side effects. A number of cyclic myelin peptide analogs seem to be potential candidates in maintaining the biological function of the original peptide and effective in controlling inflammation in EAE. Additional data regarding the immunomodulating and neuroprotective effect of these much promising agents is required. Based on the data from studies on EAE models, clinical trials should also be designed in order to elucidate the impact of such APL-induced immune responses in MS disease activity. These clinical trials should carefully incorporate monitoring of both clinical, neuroimaging and immunological parameters.

N-acetyl-L-cysteine ameliorates the inflammatory disease process in experimental autoimmune encephalomyelitis in Lewis rats

We report that N-acetyl-L-cysteine (NAC) treatment blocked induction of TNF-α, IL-1β, IFN-γ and iNOS in the CNS and attenuated clinical disease in the myelin basic protein induced model of experimental allergic encephalomyelitis (EAE) in Lewis rats. Infiltration of mononuclear cells into the CNS and induction of inflammatory cytokines and iNOS in multiple sclerosis (MS) and EAE have been implicated in subsequent disease progression and pathogenesis. To understand the mechanism of efficacy of NAC against EAE, we examined its effect on the production of cytokines and the infiltration of inflammatory cells into the CNS. NAC treatment attenuated the transmigration of mononuclear cells thereby lessening the neuroinflammatory disease. Splenocytes from NAC-treated EAE animals showed reduced IFN-γ production, a Th1 cytokine and increased IL-10 production, an anti-inflammatory cytokine. Further, splenocytes from NAC-treated EAE animals also showed decreased nitrite production when stimulated in vitro by LPS. These observations indicate that NAC treatment may be of therapeutic value in MS against the inflammatory disease process associated with the infiltration of activated mononuclear cells into the CNS.

960. Gene-Based Delivery of Interferon-beta Is Efficacious in a Murine Model of Acute Experimental Allergic Encephalomyelitis

Experimental Allergic Encephalomyelitis (EAE) is a model of central nervous system (CNS) inflammation that ensues following immunization with certain CNS antigens, for example brain derived proteolipid or myelin basic protein. The course and clinical manifestations of EAE are similar to multiple sclerosis (MS) in humans and it has become an accepted animal model to study MS. There have been several reports in which Interferon-beta (IFN-b) protein has been shown to be effective in murine and rat models of EAE. It has also been shown that local CNS delivery of a plasmid DNA encoding IFN-b decreased the severity of the disease. The purpose of this study was to demonstrate that systemic expression of murine IFN-b (mIFN-b) following gene-based delivery of plasmid DNA to hind limb skeletal muscle is effective in reducing the clinical manifestations of disease in a rodent EAE model.

C5b‐9 complement complex in autoimmune demyelination and multiple sclerosis: Dual role in neuroinflammation and neuroprotection

Complement system activation plays an important role in innate and acquired immunity. Activation of complement leads to the formation of C5b‐9 terminal complex. While C5b‐9 can promote cell lysis, sublytic assembly of C5b‐9 on plasma membranes induces cell cycle activation and survival. Multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE) are inflammatory demyelinating diseases of the central nervous system (CNS) mediated by activated lymphocytes, macrophages/microglia and the complement system. Complement activation may contribute to the pathogenesis of these diseases through its dual role: the ability of activated terminal complex C5b‐9 to promote demyelination and the capacity of sublytic C5b‐9 to protect oligodendrocytes (OLG) from apoptosis. By inducing EAE in C5‐deficient mice, we showed that complement C5 promotes remyelination and protects oligodendrocytes from apoptotic cell death. These findings indicate that activation of complement C5b‐9 plays a pro‐inflammatory role in the acute phase of the disease, but may also be neuroprotective during the chronic phase of the disease.

High-Affinity Small Molecule Inhibitors of T Cell Costimulation: Compounds for Immunotherapy

Costimulatory molecules are important regulators of T cell activation and thus favored targets for therapeutic manipulation of immune responses. One of the key costimulatory receptors is CD80, which binds the T cell ligands, CD28, and CTLA-4. We describe a set of small compounds that bind with high specificity and low nanomolar affinity to CD80. The compounds have relatively slow off-rates and block both CD28 and CTLA-4 binding, implying that they occlude the shared ligand binding site. The compounds inhibit proinflammatory cytokine release in T cell assays with submicromolar potency, and as such, they represent promising leads for the development of novel therapeutics for immune-mediated inflammatory disease. Our results also suggest that other predominantly beta proteins, such as those that dominate the cell surface, may also be accessible as potentially therapeutic targets.

Intergenomic consensus in multifactorial inheritance loci: the case of multiple sclerosis

Genetic linkage and association studies define chromosomal regions, quantitative trait loci (QTLs), which influence the phenotype of polygenic diseases. Here, we describe a global approach to determine intergenomic consensus of those regions in order to fine map QTLs and select particularly promising candidate genes for disease susceptibility or other polygenic traits. Exemplarily, human multiple sclerosis (MS) susceptibility regions were compared for sequence similarity with mouse and rat QTLs in its animal model experimental allergic encephalomyelitis (EAE). The number of intergenomic MS/EAE consensus genes (295) is significantly higher than expected if the animal model was unrelated to the human disease. Hence, this approach contributes to the empirical evaluation of animal models for their applicability to the study of human diseases.

Specific antigen vaccination to treat autoimmune disease.

Specific antigen vaccination by administration of the target antigen in aqueous solution has resulted in significant decreases of disease severity in animal models of experimental allergic encephalomyelitis, type I diabetes, and several forms of antigen-induced arthritis, even if administered after the initiation of symptoms. However, in experimental autoimmune encephalomyelitis (EAE) and type I diabetes in nonobese diabetic (NOD) mice, repeated administration of peptide fragments of target antigens in incomplete Freund's adjuvant has resulted in severe anaphylactic reactions. Although these methods of administration are known to potentiate CD4 T helper 2 (Th2) responses, which is the goal of specific antigen vaccination, the risk of anaphylaxis raises a red flag concerning use of this therapy for diseases such as type I diabetes, where the survival time after onset is quite long. It is clear that specific antigen vaccination is effective in preventing several animal models of autoimmune disease, and in treating these diseases once the symptoms are overt. However, the risks of this therapy require serious consideration of alternative methods for down-regulation of the autoimmune process.