Influence of Various Immunomodulators on the Induction of Experimental Autoimmune Encephalomyelitis in Lewis Rats

The effect of various immunomodulators on the induction of experimental autoimmune encephalomyelitis (EAE) is evaluated in the Lewis rat. Bordetella pertussis (BP) is the optimal inductor of EAE in this rat strain. Treatment of the animals with BP either before or after or simultaneously with guinea-pig spinal cord preparation (GpSC) resulted in an EAE about two weeks thereafter. Additional injection of living BCG, of CFA, IFA (incomplete Freund's adjuvant) or Vibrio cholerae neuraminidase (VCN) did not augment or mitigate the effect induced by BP or GpSC. Living BCG, IFA, VCN or Corynebacterium parvum (CP) did not induce EAE when given in combination with GpSC but without BP. CFA combined with GpSC only occasionally induced EAE. However, EAE could be induced by the combination of CFA and GpSC or IFA and GpSC in a part of the animals tested if they had been pretreated or simultaneously been injected with living BCG by intravenous route. EAE could not be enhanced by the additional injection of VCN. Surprisingly, most of the animals peracutely died after injection of CFA and BP in combination with GpSC when they had been pretreated with CP. This effect was most pronounced when pretreatment was done on day -4. No acute effect could be seen when CP was given simultaneously to CFA, BP and GpSC. Animals which did not peracutely succumb developed EAE similarly as those in the positive control groups. CP treatment simultaneously with BP but without CFA resulted in a reduction of the EAE specific mortality. This reduction could not be seen if treatment with CP was done after injection of GpSC and BP.     

Increased Expression of Phospholipase D in the Heart with Experimental Autoimmune Myocarditis in Lewis Rats

The expression of phospholipase D (PLD) in the hearts of rats with experimental autoimmune myocarditis (EAM) was studied to elucidate the functional role of PLD in the pathogenesis of EAM. Western blot analysis showed that the level of the PLD1 isoform was significantly increased in the hearts of rats with EAM on days 14, 17 and 21 postimmunization (pi) (P < 0.01; control vs EAM at 14 pi, 17 pi and 21 pi). The phenotypes of cells exhibiting increased PLD1 expression were primarily inflammatory cells, including ED1 positive macrophages, in the inflammatory EAM lesions. Some cardiomyocytes also showed increased PLD1 immunoreaction in and around EAM lesions. Some PLD1‐positive cells were also positive for proliferating cell nuclear antigen in some cardiomyocytes or terminal deoxynucleotidyl transferase (TdT)‐mediated dUTP nick end‐labeling in some macrophages, suggesting that PLD1 positive cells have a capacity for proliferation or apoptosis depending on cell types in the target organ. Thus, it is postulated that increased expression of PLD1 in EAM may support an early inflammatory response in proliferating inflammatory cells, and its expression in cardiomyocytes may help them to survive by activation of survival factors in hearts with EAM.     

Susceptibility of Lewis Rats to Experimental Autoimmune Encephalomyelitis After Recovery from Passively Induced Disease

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by adoptive transfer of 5 × 10⁷ sensitized syngeneic lymphoid cells from donors challenged 12 to 43 days earlier with myelin basic protein and complete Freund's adjuvant. The donor cells were cultured for 72 hours with antigen prior to transfer. Following recovery, the recipients remained susceptible to EAE induced by challenge with antigen and adjuvant. The relevance of this finding to host recovery mechanisms is discussed.     

Susceptibility of Lewis Rats to Experimental Autoimmune Encephalomyelitis After Recovery from Passively Induced Disease

Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats by adoptive transfer of 5 × 10⁷ sensitized syngeneic lymphoid cells from donors challenged 12 to 43 days earlier with myelin basic protein and complete Freund's adjuvant. The donor cells were cultured for 72 hours with antigen prior to transfer. Following recovery, the recipients remained susceptible to EAE induced by challenge with antigen and adjuvant. The relevance of this finding to host recovery mechanisms is discussed.     

Ia-Expressing Cells and T Lymphocytes of Different Subsets in Peripheral Nerve Tissue during Experimental Allergic Neuritis in Lewis Rats

Inflammatory infiltrates in sciatic nerves during the acute phase of experimental allergic neuritis in the Lewis rat have been characterized with regard to occurrence and distribution of Ia-expressing cells and T-lymphocyte subsets by the help of an immunohistochemical double-staining technique, enabling the simultaneous visualization of T lymphocytes and Ia-expressing non-T cells. Large numbers of Ia-expressing irregular macrophage-like/dendritic cells were seen both within inflammatory infiltrates and within afflicted nervous tissue. Many W3/13-reactive T lymphocytes of both 'helper' and 'suppressor/cytoxic' phenotypes appeared in close contact with these Ia-expressing non-T cells, particularly within the infiltrates. B lymphocytes and plasma cells were relatively few and mainly found close to endoneurial vessels.     

Berberine Ameliorates Experimental Autoimmune Neuritis by Suppressing both Cellular and Humoral Immunity

Berberine (BBR), an isoquinoline derivative alkaloid, has been extensively used in traditional Chinese medicine for the treatment of diarrhoea, rheumatic diseases, diabetes, etc. Recent studies have demonstrated new biological properties of BBR and suggested the possibility of BBR to be a therapeutic agent for some autoimmune diseases. To explore the effect of BBR on the development of experimental autoimmune neuritis (EAN), BBR was administered intragastrically daily to Lewis rats immunized with P0 peptide 180–199 in Freund's complete adjuvant. We found BBR treatment resulted in amelioration of EAN, accompanied by suppressed lymphocyte (in particular CD4⁺ T cell) proliferation, downregulated Th1 (TNF‐α) and Th2 (IL‐10) cytokines and reduced anti‐P0 peptide 180–199 IgG1 and IgG2a. In brief, BBR played a role in ameliorating EAN by suppressing both cellular and humoral immunity. Thus, our study suggests that BBR may be a potential therapeutic agent for the autoimmune disease in the peripheral nervous system, such as Guillain–Barré syndrome.     

Molsidomine Ameliorates Experimental Allergic Encephalomyelitis in Lewis Rats

Experimental allergic encephalomyelitis (EAE) is an autoimmune CD4⁺ T cell-mediated disease of the central nervous system (CNS). Nitric oxide (NO) plays an important role in preventing the development of EAE. Molsidomine (Mol) is a drug used for the treatment of coronary artery disease. Its therapeutic effects are the consequences of NO formation. In this study, we investigated the effects of Mol on EAE development in myelin basic protein (MBP)-immunized Lewis rats. All rats immunized with MBP developed typical clinical signs of acute EAE. In the EAE rats receiving Mol, the severity of clinical signs and the infiltration of inflammatory cells in CNS were clearly reduced. Furthermore, Mol administration significantly reduced the production of interferon-γ, a Th1 inflammatory cytokine, but increased the production of interleukin-10, a Th2 anti-inflammatory cytokine. Our findings suggest that the administration of the exogenous NO donor Mol is of considerable benefit in limiting the development of EAE and other Th1 cell-mediated inflammatory diseases.     

T-Cell Immunity to Acetylcholine Receptor and its Subunits in Lewis Rats over the Course of Experimental Autoimmune Myasthenia Gravis

Lymph nodes, spleen and thymus obtained from Lewis rats were examined over the course of experimental autoimmune myasthenia gravis (EAMG) for the distribution and the number of antigen-reactive CD4⁺ T helper cells which, upon recognition of Torpedo acetylcholine receptor (AChR) or the α, β, γ or δ subunits of Torpedo AChR, responded by secretion of interferon-gamma (IFN-γ). T cells with these specificities were detected in these three immune organs. Numbers were highest in lymph nodes. In spleen and thymus, numbers of antigen-reactive T cells did not differ. T cells reacting against the intact AChR were more frequent than T cells recognizing any of the subunits. The immunogenicity between the four subunits did not differ, with the exception that the α subunit induced a slightly higher T-cell response. No restriction of the T-cell repertoire to the four subunits was detected during early compared to late phases of EAMG. The AChR and subunit-reactive T cells could—via secretion of effector molecules including IFN-γ—play an important role in the initiation and perpetuation of EAMG. and consequently also of human myasthenia gravis. T cells with the same specificities were also detected in control animals injected with adjuvant only, but at much lower numbers which were within the range of T cells recognizing the control antigen myelin basic protein. They could represent naturally occurring autoimmune T cells.     

Multiple Autoantigen Mimotopes of Infectious Agents Induce Autoimmune Arthritis and Uveitis in Lewis Rats

We found multimolecular antigenic mimicry of arthritogenic autoantigens and peptides from several other “self” or foreign antigens sharing amino acid sequence homologies. Many of these new mimotopes induced arthritis and/or uveitis upon immunization in Lewis rats, indicating a role for multiple antigens in the initiation of a certain autoimmune disease.     

Propentofylline and Iloprost Suppress the Production of TNF-αby Macrophages but Fail to Ameliorate Experimental Autoimmune Encephalomyelitis in Lewis Rats

Intracellular cAMP levels can be elevated by activation of cAMP-generating adenylate cyclase (AC) or inhibition of cAMP-cleavage by phosphodiesterases. Elevation of intracellular cAMP levels in immune cells inhibits production of some Th1-cytokines, particularly TNF-α, and results mainly in downregu-lation of the immune response. Experimental autoimmune encephalomyelitis (EAE) of Lewis rats is a disease mediated by type 1 T helper lymphocytes and macrophages and serves as a model of multiple sclerosis. In EAE we therefore tested the immunomodulatory potency of an AC-activating, stable prosta-cyclin analogue, iloprost, and of a potent and non-selective inhibitor of phosphodiesterases, propentofylline, which also has neuroprotective properties.Preventive treatment of Lewis rats with propentofylline (2×10 or 12.5 mg/kg/d), iloprost (2×10 or 12.5 μg/kg/d), or both did not significantly ameliorate clinical or histological signs of EAE actively induced by immunization with myelin basic protein (MBP) in complete Freund's adjuvant. Furthermore, adoptive transfer EAE (AT-EAE), passively induced by injection of encephalitogenic MBP-specific Th1 lymphocytes, was not altered in its course by the combined application of iloprost (2×10 μg/kg/d) and propentofylline (2×20 mg/kg/d) starting on the day of cell transfer. In vitro assays demonstrated that iloprost strongly and propentofylline moderately inhibited the production of TNF-α by macrophages and that iloprost in vivo similarly suppressed TNF-α secretion, although this effect was limited to a few hours after a single injection. In contrast to macrophages, TNF-α production by antigen-activated encephalitogenic T helper line cells in vitro was completely resistant to modulation by these agents. In addition, the presence of iloprost, propentofylline, or both drugs during activation of the line cells in vitro did not impair their encephalitogenicity in vivo.The findings delineate immunomodulatory effects of both substances, particularly of iloprost, but fail to support a possible therapeutic role of these agents in autoimmune inflammation of the central nervous system.     

Mitigated Tregs and Augmented Th17 Cells and Cytokines are Associated with Severity of Experimental Autoimmune Neuritis

Experimental autoimmune neuritis (EAN), an animal model of human Guillain–Barré syndrome, has long been considered as a T helper (Th) 1 cell–mediated autoimmune disorder. However, deficiency of IFN‐γ, a signature Th1 cytokine, aggravated EAN, with features of elevated production of IL‐17A, despite an alleviated systemic Th1 immune response. We hypothesized that Th17 cells and their cytokines might play a pathogenic role in EAN. To further clarify the roles of these Th and regulatory T cell (Treg) cytokines in the pathogenesis of EAN and their interrelationship, we investigated the expression of Th1/Th2/Th17/Treg cytokines in EAN in this study. We found that the levels of Th17 cells and IL‐17A in cauda equina (CE)‐infiltrating cells and splenic mononuclear cells (MNCs) as well as in serum paralleled the disease evolution, which increased progressively during the initiation stage and reached higher value at the peak of EAN. The same pattern was also noticed for the expression of IL‐22. The diverse expression profiles of FoxP3, IL‐17 receptors A and C were seen in CE‐infiltrating cells and splenic MNCs in EAN. These findings indicate a major pro‐inflammatory role of Th17 cells and IL‐17A in the pathogenesis of EAN. Therapeutic interventions may be focused upon inhibiting Th17 cells and their cytokines in the early phase of EAN, so as to delay and suppress clinical signs of the disease, which has relevance for future studies on pathogenesis and treatment of GBS in humans.     

重肌灵片对实验性重症肌无力的影响

用电鳐电器官提取的乙酰胆碱受体粗提物与完全福氏佐剂等量混合多点免疫Lewis大鼠制备EAMG大鼠模型,研究中药重肌灵片的作用,发现重肌灵片两个剂量组(6.56 g/kg和3.28 g/kg)均能显著改善EAMG大鼠临床症状,缓解连续低频电刺激所致大鼠后肢肌肉收缩幅度的衰减,降低血清中抗乙酰胆碱受体抗体的含量,抑制乙酰胆碱受体特异性的淋巴细胞增殖并能降低突触后膜上乙酰胆碱受体的减少。提示重肌灵能有效地改善EAMG大鼠临床症状及实验室指标。     

Proteomic Profiling Analysis Reveals a Link between Experimental Autoimmune Uveitis and Complement Activation in Rats

Uveitis is an autoimmune disease that usually damages the vision function, leading to poor visual quality in patients. As an autoimmune ocular inflammatory disease, the pathogenesis of uveitis is associated with abnormal expression of some proteins and aberrant regulation of multiple signalling pathways. Nevertheless, the detailed mechanism remains unclear. In this study, we induced an experimental autoimmune uveitis (EAU) model in rats. We determined the levels of C3a and membrane attack complex C5b‐9 (soluble C5b‐9, sC5b‐9) in both plasma and aqueous humour, identified the differentially expressed proteins in plasma by liquid chromatography–tandem mass spectrometry and employed bioinformatics algorithms to analyse differentially expressed proteins in EAU rat plasma. The results demonstrate that there were 168 differentially expressed plasma proteins in EAU rats versus control subjects. The levels of sC5b‐9 and C3a were elevated in the plasmas and aqueous humours of EAU rats. Gene ontology enrichment analysis showed that the differentially expressed proteins in EAU rat plasma were mainly involved in metabolic and immune processes. Kyoto encyclopedia of genes and genomes (KEGG) pathway annotation, database for annotation, visualization and integrated discovery (DAVID) and protein–protein interaction analyses revealed that the differentially expressed proteins in EAU rat plasmas were closely associated with complement and coagulation cascades, metabolic pathways, NF‐kappa B, PI3K‐Akt, Toll‐like receptors and autophagy. Overall, the differentially expressed proteins in EAU rat plasmas are mainly involved in the complement and coagulation cascades. The pathogenesis of uveitis closely correlates with complement activation.     

The Strategies Used for Treatment of Experimental Autoimmune Neuritis (EAN): A Beneficial Effect of Glatiramer Acetate Administered Intraperitoneally

Glatiramer acetate (GA) significantly ameliorates multiple sclerosis and was initially discovered through its effects on the animal model experimental autoimmune encephalomyelitis (EAE). Guillain-Barré syndrome (GBS) is a relatively common demyelinating disease of peripheral nerves for which there is a parallel animal model, experimental autoimmune neuritis (EAN). We review the treatments found useful in EAN with special emphasis on the need for quick onset of action and the relevance of treatments used for EAE and multiple sclerosis. We evaluated the effect of GA administered by a novel intraperitoneal route in EAN. GA significantly ameliorated the severity of disease in rats (F = 6.3, p = 0.01 by analysis of variance (ANOVA)) and course of disease (F = 4.9, p = 0.02 by repeated-measures ANOVA with a day × treatment interaction term). Neurophysiology data supported the trend for the beneficial effect of GA. Myelin-induced immune cell proliferation was significantly modulated by GA (p < 0.025). This report describes a novel route of administration of GA and a rapid beneficial effect of GA in EAN. GA may be useful in human diseases, such as GBS, where the intravenous route may offer a rapid onset of drug action.     

Gene Therapy in Experimental Autoimmune Encephalomyelitis

Gene therapy traditionally has been associated with gene replacement, where exogenous recombinant DNA is introduced ex vivo into somatic cells that are then introduced back into the patient as a way to correct an inherited genetic defect. However, several novel gene therapy strategies for treating autoimmune diseases recently have emerged. Strategies involving the use of several types of DNA vaccines, the application of various viral vectors, and the use of diverse cellular vectors have shown promise in inhibiting autoimmune-mediated inflammation and repairing tissue damaged as a result of autoimmune attack. In the current review, we examine and discuss the development and proposed use of emerging gene therapy strategies for the treatment of autoimmune disease with specific emphasis on experimental autoimmune encephalomyelitis (EAE), an animal model widely used in multiple sclerosis (MS) research.     

Antibody Response to Myelin Basic Protein: Comparisons Between Lewis Rats with Experimental Allergic Encephalomyelitis, and Tolerant Rats

The hemagglutinating antibody responses of Lewis rats with experimental allergic encephalomyelitis (EAE) and of rats rendered unresponsive to this autoimmune disease by pretreatment with myelin basic protein (BP) were compared. Most tolerant animals produced low levels of hemagglutinating antibody. Similarly, most rats with EAE also produced anti-BP antibodies. We were unable to correlate hemagglutinin production or titer with protection against disease. Hemagglutination inhibition (HAI) studies reveal cross-reactivity between rat, guinea pig and bovine BP. HAI studies with BP-derived peptides suggest that at least three distinct antibody-binding determinants exist in the BP molecule, and that individual inbred Lewis rats respond differently with respect to antibody production to these sites.     

Modulation of Experimental Allergic Encephalomyelitis in Lewis Rats by Administration of a Peptide of Fas Ligand

The effects of modulation of apoptosis in experimental allergic encephalomyelitis (EAE) in Lewis rats have been investigated using a peptide of the Fas-Ligand protein (FasL-p). The peptide was administered both subcutaneously and intra-cerebro-ventricularly (i.c.v.) after EAE induction. Rats treated subcutaneously with FasL-p showed a worse clinical score as compared to saline treated animals, while i.c.v. treatment with FasL-p did not modify significantly the severity of EAE. Apoptotic lymphomonocytes (identified by TUNEL) infiltrating the brain and the spinal cord were decreased in rats treated i.c.v. with FasL-p. The data suggest that the Fas/Fas-ligand pathway may be modulated by treatments with peptides of Fas-Ligand and that it may be at work within the central nervous system in EAE.     

Modeled Microgravity Suppressed Expansion of the MBP-specific T Lymphocytes of Rats with Experimental Autoimmune Encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) is characterized by uncontrolled proliferation of autoreactive T lymphocytes, with markedly increased secretion of pro-inflammatory cytokines. To further dissect the pathogenetic pathways of this disease, we exposed T lymphocytes from EAE rats, which were specific for myelin basic protein (MBP) to a modeled microgravity (MMG) environment, using a rotated cell culture system (RCCS) that was known to suppress proliferation of normal T cells. Following exposure to MMG, the proliferation of EAE lymphocytes decreased dramatically compared to those cultured in unit gravity (UG). At the beginning of MMG, a significant increase of apoptosis of MBP-specific T lymphocytes was observed, while at a later stage, the cytokine secretion profile of exposed MBP-specific T lymphocytes was altered, as was the differentiation of Th subsets. We concluded that the function of MBP-specific T lymphocytes was disordered after exposure to MMG.     

Interaction between Gonadal Steroids and Neuroimmune System in Acute Experimental Autoimmune Encephalomyelitis (EAE) in Wistar Rats

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory disease of the CNS mediated by autoreactive T lymphocytes directed against myelin antigens. Since neuroendocrine-immune dysfunction appears to contribute to the pathogenesis of autoimmune diseases, the present work was designed to study the effect of changes in the endocrine system on the development of acute EAE and the immune response against myelin basic protein (MBP). Intact and sham males and intact female Wistar rats showed the most severe clinical symptoms (acute period) 12–14 days post-inoculation (dpi). Then, they began gradually to recover, regaining the total ability to walk by 15–17 dpi. Male Wistar rats with altered levels of gonadal hormones by surgical castration showed an onset of the symptoms retarded 2–3 days with respect to the other EAE groups, showing neuropathological symptoms up to 27–28 dpi, and remaining with lower body weight even at 40 dpi. The castrated animals exhibited a specific delay in MBP-stimulated DTH reactivity that correlates with the delay in the onset of the clinical symptoms. Also significant lymphocyte proliferation to MBP was still present at 35 dpi that was absent in the sham group. The distribution of the IgG subclasses indicated that at 35 dpi castrated animals have a higher IgG2b/IgG1 ratio (35.1) in comparison to that presented by sham rats (4.8). Considering that at this time the castrated animals were not completely recuperated, these results could indicate an ongoing inflammatory immune response associated with Th1 activity in these animals. Also castrated animals developed antibodies to a diversity of MBP epitopes in comparison to sham rats, which presented a dominance of antibodies to MBP peptide p96–128. These results indicate that sex hormones levels regulate cell-mediated immunity and the specificity of anti-MBP antibodies related to the induction and development of acute EAE.