Sympathectomy augments adoptively transferred experimental allergic encephalomyelitis

Adoptively transferred experimental allergic encephalomyelitis (EAE) was significantly augmented in Lewis rats with ablated sympathetic nervous system. Sympathectomy was obtained by treatment of newborn rats with 6-hydroxydopamine. Sham-injected rats were used as a control. EAE was elicited in 7-8-week-old donor Lewis rats by immunization with a suspension of guinea pig (GP) brain and spinal cord in complete Freund's adjuvant. Successful transfer of EAE was accomplished with 50 x 10(6) lymph node cells (LNC)/rat, incubated for 72 h with GP myelin basic protein. LNC were obtained from draining lymph nodes, 9 days after immunization for EAE. The severity of passively transferred EAE was significantly augmented when donor LNC obtained from normal Lewis rats immunized for EAE were injected into sympathectomized rats as compared to sham-injected rats. When LNC were obtained from sympathectomized or sham-injected donors, the disease was significantly more severe in recipients of cells from sympathectomized animals. The severity of histological lesions in the brain and spinal cord was greater in rats with passively transferred EAE which received LNC from sympathectomized donors.     

Interferon-gamma inhibition suppresses experimental allergic neuritis: modulation of major histocompatibility complex expression of Schwann cells in vitro

This study examines the modulation of major histocompatibility complex (MHC) expression on Lewis rat Schwann cells (Scs) cultured in the presence of dorsal root ganglion neurons (DRG). MHC class I and II molecules were induced on Scs using recombinant murine interferon-gamma (IFN-gamma), lymph node cells (LNC), removed at day 9 from Lewis rats with experimental allergic neuritis (EAN) and syngeneic T-cell line cells responsive to P2 basic protein. EAN LNC induced MHC class I on Scs but only IFN-gamma or P2-responsive T-cells induced MHC class II. Control LNC from animals injected with Freund's adjuvant alone or naive spleen cells did not induce MHC class II. P2 T-cells clustered in aggregates to the Scs. Similar studies were performed with inhibitors of IFN-gamma; hydrocortisone, cyclosporin A, dibutyryl cyclic AMP, methyl-xanthine and prostaglandin E2. Each agent produced a dose-dependent inhibition of MHC expression and prevented clustering of P2-responsive T-cells to Scs.     

Induction of experimental autoimmune neuritis in CD4-8-C57BL/6J mice.

The C57BL/6J mice strain is known to be reputedly resistant to induction of experimental autoimmune neuritis (EAN), an animal model of Guillain-Barré syndrome in humans. Here we describe the induction of EAN in mice of the C57BL/6J background by transfer into naive syngeneic recipients bovine peripheral nerve myelin (BPM)-primed donor lymph node cells that had been stimulated in vitro with the bovine peripheral nervous system (PNS) myelin P2 protein peptide 57-81 followed by challenge with BPM, Freund's complete adjuvant and pertussis toxin. EAN was more severe, both clinically and histologically, and accompanied by extensive infiltration of inflammatory cells and demyelination in peripheral nerves when examined on day 30 after transfer of primed T cells from CD4- 8- mice into identical naive hosts than after transfer of cells from primed wild type, CD4-/- or CD8-/- mice to corresponding recipient animals. EAN in CD4-8- mice was also associated with elevated numbers of P2 peptide-reactive interferon-y (TFN-gamma) secreting cells and alphabeta T cells were present in lymph nodes and spleens. The data suggest that PNS myelin activated T cells from an EAN-resistant mice strain are capable of homing to the PNS. The expanded CD4-8- alphabeta T cells may have helper and effector functions, related to initiation of EAN in the CD4-8- mice. Lack of CD4+ and CD8+ expressing cells does not prevent the initiation of an autoimmune disease.     

Immune responses in experimental allergic neuritis.

The antibody and cell mediated immune responses were investigated in inbred Lewis rats with experimental allergic neuritis (EAN) induced by either P2, a protein purified from the bovine cauda equina nerve roots, or whole bovine nerve root myelin. In the P2 immunised animals both antibodies to P2 detected by radioimmunoassay and cell-mediated immunity to P2 assayed by skin testing appeared before the onset of EAN and persisted during and after the disease. In the myelin immunised animals the antibody titres were lower and somewhat delayed and the skin tests became negative at the height of the disease. Complement-fixing antibodies to galactocerebroside, which have been implicated in the production of demyelination under some circumstances, could not be detected in the serum after immunisation with either P2 or myelin. EAN was transferred passively with lymph node cells from rats immunised with either P2 or myelin although anti-P2 antibodies could not be detected in the serum of recipients with EAN. The results favour a cell-mediated immune response to P2 as the most important pathogenetic mechanism in EAN induced wtih whole myelin in the rat.     

Adoptive transfer of experimental allergic encephalomyelitis: Recipient response to myelin basic protein-reactive lymphocytes

We have used adoptive transfer of myelin basic protein (MBP)-reactive lymphocytes in the Lewis rat model of experimental allergic encephalomyelitis (EAE) to identify stages of effector cell development and to investigate the nature of the subsequent recipient response to the transferred cells. Depending on the timing of cell collection, lymph node cells (LNC) obtained from MBP-CFA (MBP emulsified in complete Freund's adjuvant)-immunized donors may directly transfer clinical disease; however, independent of disease development, recipients of LNC develop early onset of clinical disease following immunization of the recipients with MBP-CFA, consistent with the presence of MBP-memory cells in the LNC transfer inoculum. Similarly obtained spleen cells do not directly transfer disease and do not contain MBP-memory cells (as defined by the early onset of clinical disease following MBP-CFA challenge). Spleen cells adoptively transfer clinical disease only following in vitro culture stimulation with antigen or selected mitogens. Recipients of the primary culture-derived encephalitogenic spleen cells also develop an accelerated onset of clinical disease following MBP-CFA challenge, indicative of the presence of MBP-memory cells, and are not vaccinated. Encephalitogenic T cell lines adoptively transfer clinical disease, and in most cases recipients are vaccinated to MBP-CFA-induced active disease, but remain susceptible to adoptively transferred disease. Co-transfer of encephalitogenic T cell line cells with MBP-reactive lymph node or encephalitogenic spleen cells does not alter the vaccination response. We have found that during the process of T cell line development, the vaccinating phenotype is acquired following the second antigen stimulation cycle. These studies also demonstrate that regulation induced by T cell vaccination blocks the development of effector cells from precursor cells and that such regulation is also equally effective in blocking disease development in recipients which have increased numbers of memory cells. Thus, the response to T cell vaccination, once established, is fully capable of inhibiting the development of effector cells from increased numbers of precursor/memory cells, a response that would be needed in the clinical application of vaccination-induced resistance.     

The role of myelin P2 protein in the production of experimental allergic neuritis

Myelin P2 protein has been proposed as the primary antigen in whole myelin-induced experimental allergic neuritis (EAN). We investigated the neuritogenic properties of P2 by sensitizing Lewis rats with complete Freund's adjuvant (CFA) containing P2, P2 plus phosphatidyl serine, or whole myelin containing an equivalent amount of P2. Animals were examined using a battery of clinical, electrophysiological, immunological, and morphological methods. Myelin-immunized rats developed the characteristic features of EAN. P2-sensitized rats developed a similar but much less intense disorder. When rats were sensitized with P2 in the presence of phosphatidyl serine, however, they developed radiculoneuropathy that was indistinguishable from myelin-induced EAN. Inoculation with phosphatidyl serine plus complete Freund's adjuvant or complete Freund's adjuvant alone had no detectable effect on peripheral nerves. These studies demonstrate that sensitization of rats with a single myelin antigen, P2 protein, is sufficient to induce the clinical, electrophysiological, and neuropathological features of EAN.     

Chronic relapsing experimental allergic encephalomyelitis in the Lewis rat: studies on immune regulation

To study immunoregulation of chronic relapsing experimental allergic encephalomyelitis (CR-EAE) in Lewis rats, we adoptively transferred concanavalin A-activated lymph node cells (LNC) or splenocytes, from hind footpad-inoculated donors at the onset (day 11), or recovery (day 16), of the first attack. Popliteal LNC, especially from day 16 donors, provided significant and dose-dependent, but incomplete, protection of recipients from encephalitogenic challenge; maximal mean delay in EAE onset was 10 days later than controls, with subsequent paralysis reduced more than 6-fold. In contrast, particularly from day 11 donors, superficial inguinal LNC recipients developed actively induced disease of normal severity up to 4 days earlier than CR-EAE controls. Furthermore day 11 EAE splenocytes, but not day 16 ones, adoptively transferred disease into 50-88% of naive recipients. In separate studies, we demonstrated unresponsiveness to active induction of disease in all rats re-challenged during stable late remission, as well as in a minority of animals pretreated with antigen in incomplete Freund's adjuvant. These results suggest an organ-dependent and time-dependent balance between effector and suppressor populations in the model.     

T cell vaccination does not induce resistance to experimental autoimmune neuritis.

The effectiveness of T cell vaccination was analyzed in experimental autoimmune neuritis (EAN) that can be induced by immunization with bovine P2 protein or a peptide representing the amino acids 53-78 of P2 (P2 53-78). Lewis rats were vaccinated with glutaraldehyde-fixed lymph node cells which had been primed in vivo with P2 protein or P2 53-78 and had been activated in vitro with concanavalin A. Vaccinated animals were not protected from EAN induced by immunization with P2 protein in complete Freund's adjuvant (CFA). In a second set of experiments Lewis rats were vaccinated with irradiated or fixed P2-specific T cell lines of different specificity and neuritogenicity and were subsequently challenged with P2 53-78 in CFA. Likewise, severity of P2 53-78-induced EAN was not different between naive and T line-vaccinated groups. In spleens of vaccinated animals a substantial suppressive activity was demonstrated which was positively correlated with a weak anti-ergotypic response of these spleen cells. The fact that development of actively induced EAN was not prevented or even mitigated by T cell vaccination, in spite of an apparent vaccination-induced response to and on T lymphocytes, suggests that protection from disease is not readily induced in every autoimmune disease model.     

Enhancement of acute phase and inhibition of chronic phase of experimental autoimmune neuritis in Lewis rats by intranasal administration of recombinant mouse interleukin 17: potential immunoregulatory role

Experimental autoimmune neuritis (EAN) is a CD4(+) T-cell-mediated demyelinating disease of the peripheral nervous system (PNS). We examined the effect of recombinant mouse interleukin 17 (rmIL-17) on chronic EAN induced in Lewis rats by inoculation of P2 57-81 peptide in Freund's complete adjuvant. Animals were treated nasally for 6 days with either 0.1 or 0.9 microg/rat/day rmIL-17 from the onset of neurological signs, i.e., days 9 to 14 postimmunization (p.i.). Prolonged follow-up demonstrated a chronic course in control and rmIL-17-treated rats. Treated rats had more severe disease initially (days 18-36 p.i.) with a stronger enhancing effect observed with the higher rmIL-17 dose. At day 19 rmIL-17-treated rats showed increased infiltration of inflammatory cells into the sciatic nerve, more severe demyelination, augmented proliferation of regional lymph node cells, and increased serum levels of tumor necrosis factor-alpha. After the initial phase of disease enhancement the IL-17-treated EAN rats improved gradually and ultimately recovered completely, whereas the control EAN rats remained affected until the end of the observation (day 120 p.i.). The lower dose of rmIL-17 induced an earlier recovery from clinical deficits than the higher one. The results indicate that IL-17 plays an immunoregulatory role in chronic EAN which could have implications for immunomodulatory treatments of chronic autoimmune disease of the PNS.     

Spontaneous chemiluminescence activity of peripheral blood cells during experimental allergic neuritis (EAN)

In 30 Lewis rats the time course of spontaneous monocyte and granulocyte chemiluminescence activity (CLA) during myelin-induced experimental allergic neuritis (EAN) was studied. CLA of blood and peritoneal cells, and clinical, histopathological and electrophysiological findings were compared with data from 14 animals immunized with complete Freund's adjuvant and ten normal Lewis rats. About 12 days post-immunization there was a significant (p less than 0.003) rise in monocyte, but not in granulocyte CLA in EAN animals. CLA correlated significantly (p less than 0.02) with infiltrate formation in nerve roots, preceding clinical or electrophysiological signs of disease by 1-2 days. There was no increase in peritoneal monocyte CLA, which was consistently higher than blood monocyte CLA (p less than 0.002).     

Diurnal rhythms in ornithine decarboxylase activity and norepinephrine and acetylcholine synthesis in submaxillary lymph nodes and spleen of young and aged rats during Freund''s adjuvant-induced arthritis

Aging has been associated with attenuation of amplitude and changes in period of many circadian rhythms. The present study was carried out to examine, in young (50 days old) and old (18 months old) rats, whether 24-h rhythms of cell proliferation (as assessed by measuring ornithine decarboxylase activity) and of presynaptic adrenergic and cholinergic markers change in lymph nodes and spleen during Freund's adjuvant-induced arthritis. Groups of young and old Sprague–Dawley rats were studied the day before, and on days 6, 12 and 18 after Freund's adjuvant injection. On day 16 after adjuvant injection, inflammation of hind paws, mainly in the ankle joints, was less marked in old than in young rats. Lymph node and splenic ornithine decarboxylase activity exhibited significant 24-h variations with maximal activity during daily hours. Before treatment, enzyme activity values were significantly lower in old rats in both tissues examined. During the immune reaction, lymph node and splenic ornithine decarboxylase augmented 8–10-fold, with progressively smaller amplitude of daily variations as arthritis developed. In every case, mesor and amplitude of ornithine decarboxylase activity were lowest in old rats. Submaxillary lymph node and splenic tyrosine hydroxylase activity attained maximal values at night. At every time interval after mycobacterium adjuvant injection, amplitude and mesor of tyrosine hydroxylase activity rhythm were lowest in old rats. A maximum in submaxillary lymph node ³H-acetylcholine synthesis occurred at the afternoon. On day 6 and 12 after Freund's adjuvant injection, lymph node ³H-acetylcholine synthesis was significantly smaller in old rats. Day–night differences in submaxillary lymph node or splenic ornithine decarboxylase and tyrosine hydroxylase activities, or in submaxillary lymph node ³H-acetylcholine synthesis, of rats treated with the adjuvant's vehicle, did not differ significantly from those seen in untreated controls. The results are compatible with an age-dependent decline of immune-mediated inflammatory responses. The activity of the central circadian oscillator, driven to the organs in part via the autonomic nervous system, seems also to deteriorate during aging.     

Dominance of autoreactive T cell-mediated delayed-type hypersensitivity or antibody-mediated demyelination results in distinct forms of experimental autoimmune neuritis in the Lewis rat

The role of anti-myelin antibodies in the pathogenesis of experimental autoimmune neuritis (EAN) induced in the Lewis rat by immunization with peripheral nerve myelin has been assessed. Passive transfer with lymph node cells (LNC) or purified serum immunoglobulin from rats with EAN was employed to directly measure the contribution of B cells and anti-myelin antibodies to demyelination and disease. Lewis rats with EAN transferred by LNC or purified serum immunoglobulin from EAN donors in conjunction with a low dose of P2-specific CD4+ T cells demonstrated profound histopathological and neurophysiological evidence of demyelination during disease. In contrast, the classical adoptive transfer model of EAN in the Lewis rat induced by the injection of P2-specific CD4+ T cells was characterized by histopathological and neurophysiological evidence of axonal dysfunction and degeneration with limited demyelination. These findings demonstrate that the synergistic action of T cells and anti-myelin antibodies mediating demyelination or purely T cell mediated axonal dysfunction and degeneration are distinct pathways by which a specific autoimmune response in the peripheral nervous system can cause neurological disease.     

Chronic relapsing experimental allergic neuritis in Lewis rats: Effects of thymectomy and splenectomy

Summary Experimental allergic neuritis (EAN) was induced in Lewis rats aged 4 months by the inoculation of whole bovine dorsal root with Freund's complete adjuvant. Prolonged follow-up demonstrated that a relapsing course is a regular feature of the disorder in animals at this age. Although the initial disease episode was the most severe, clinical recovery from subsequent relapses was less satisfactory, this probably being related to persistent morphological abnormalities in the peripheral nervous system. Antecedent thymectomy, splenectomy, or the two combined, had little effect on the clinical course of the disorder, apart from reducing the duration of relapses. This was only statistically significant following combined thymectomy/splenectomy. Histological abnormalities, however, tended to be less severe in the operated as compared with normal control or sham-operated animals with EAN. The animals must have attained an immunocompetent state at the time of thymectomy and/or splenectomy. The capacity to develop EAN presumably resides in the draining lymph nodes and the occurrence of relapses is due to the continuing presence of antigen at the injection sites.Supported by the National Fund for Research into Crippling Diseases and the Medical Research Council     

Regulation of experimental allergic encephalomyelitis : Part 4. Further characterization of postrecovery suppressor cells

Suppressor cells that regulate experimental allergic encephalomyelitis (EAE) are present in lymph nodes of Lewis rats that have recovered from the disease, as demonstrated by adoptive transfer of suppression for at least 108 days following challenge of donor rats with myelin basic protein (BP) in complete Freund's adjuvant (CFA). These suppressor lymph node cells (LNC) inhibit the occurrence of active, but not passive EAE in recipients, suggesting that they function early in the inductive phase of the response to BP. The suppressor cells are nylon-adherent, and suppression can be partially abrogated by depletion of B cells with anti-immunoglobulin (ig) and complement (C). Serum from recovered donors has, at best, only a modest suppressive effect when transfused into syngeneic recipients. These findings suggest that the B cells do not mediate suppression on EAE via production of blocking antibody, but may function directly, perhaps by triggering T cells to become suppressor cells.     

Rho激酶抑制剂对OX40及其配体mRNA在实验性变态反应性神经炎中表达的影响

目的 研究Rho激酶(ROK)抑制剂对OX40及其配体(OX40L)mRNA在实验性变态反应性神经炎(experimental allegic neuritis,EAN)大鼠坐骨神经、脾脏、外周血和淋巴结中表达的影响.方法 54只Lewis大鼠随机分为EAN模型组、EAN+ROK抑制剂干预组和完全弗氏佐剂对照(CFA)组.分别在免疫后第9、17、26天处死动物,取其坐骨神经根、脾脏、外周血单个核细胞和淋巴结,采用逆转录PCR(RT-PCR)技术检测OX40和OX40L mRNA在各组织的表达水平.结果 EAN+ROK抑制剂组大鼠OX40 mRNA在坐骨神经中第9、17、26天的表达分别为0.266±0.031、0.298±0.024和0.113±0.018;在淋巴结中第9、17、26天的表达分别为0.453±0.030、0.496±0.100和0.220±0.016;OX40L mRNA在坐骨神经中第9、17、26天的表达分别为0.247±0.018、0.298±0.026和0.165±0.013;在淋巴结中第9、17、26天的表达分别为0.283±0.027、0.306±0.011和0.161±0.012.与EAN组比较,OX40和OX40L mRNA的表达明显降低(t=2.24～4.89,P＜0.05),坐骨神经炎性细胞浸润和脱髓鞘减轻.CFA组大鼠无症状.结论 ROK抑制剂可以减轻EAN发病程度,抑制OX40/OX40L的表达可能是其作用机制之一.     

Passive transfer of experimental autoimmune encephalomyelitis in Wistar rats: dissociation of clinical symptoms and biochemical alterations

We have used passive transfer of myelin-reactive lymphocytes in the Wistar rat model of experimental autoimmune encephalomyelitis (EAE) to investigate the nature of the central nervous system immunopathological alterations induced by these cells. Mononuclear cells from lymph nodes or spleen from sick myelin/complete Freund's adjuvant-immunized donors did not transfer clinical disease. However, depending on the previous treatment of the transferred cells, recipients develop central nervous system biochemical and histological alterations. Fresh cells from lymph nodes immediately transferred after procurement from the sick EAE donor rat were capable of inducing the most significant diminution in the content of myelin basic protein, sulfatides, and 2',3'-cyclic nucleotide-3'-phosphohydrolase activity, with concomitant inflammatory infiltrations of white matter, principally in spinal cord and cerebellar lobules. Similar alterations were observed when animals were injected with spleen mononuclear cells activated in the presence of a nonspecific mitogen as concanavalin A. However, antigen-specific activated spleen cells generated by culturing in the presence of bovine myelin induced alterations to a lesser degree. Results point to a dissociation of the clinical disease from the central nervous system biochemical and histopathological lesions occurring in the EAE-transferred Wistar rats and indicate that these alterations in EAE are induced principally by T cells activated in vivo rather than by cells activated in vitro by myelin antigens. Therefore, these findings suggest a possible participation of lymphocytes unlike the encephalitogenic T cells in the induction of the described alterations and provide a useful model to explore further the subclinical responses to this experimental disease.     

Peripheral nerve demyelination in rabbits after inoculation with Freund's complete adjuvant alone or in combination with lipid haptens

The pathology of demyelination in rabbits with experimental allergic neuritis (EAN) or galactocerebroside-induced neuritis was compared to that in rabbits inoculated with either an emulsion of lipid haptens (gangliosides, lecithin and cholesterol) and Freund's complete adjuvant or Freund's complete adjuvant (FCA) alone. In rabbits inoculated with bovine peripheral myelin in FCA, perivenular demyelination associated with infiltrates of lymphocytes and macrophages occurred after 30 days, while those animals inoculated with galactocerebroside (GC) in Freund's adjuvant did not develop lesions until 60-90 days. GC rabbits had demyelination and severe nerve edema without cellular infiltrates. In rabbits inoculated with FCA alone, demyelination was restricted to ganglia and proximal nerve roots. Myelin basic protein (MBP) and GC antibodies from EAN, GC and lipid hapten-inoculated rabbits were detected by ELISA in sera at all post-inoculation time points. Appreciable P0 and P2 antibody titers were detected only in EAN animals. The results indicate that Freund's complete adjuvant alone or in combination with lipid haptens is capable of producing neuropathic effects in the rabbit independent of those produced by EAN or galactocerebroside neuritis.     

Passive transfer of experimental allergic encephalomyelitis induced by proteolipid apoprotein

In an attempt to obtain insight into the pathogenesis of proteolipid apoprotein (PLP)-induced experimental allergic encephalomyelitis (EAE) in Lewis rats (Yamamura et al. 1986), PLP-sensitized lymph node cells or spleen cells were passively transferred into normal or irradiated (400 rad) recipients after incubation with concanavalin A or PLP. Clinical EAE manifested by paraparesis was successfully transferred into irradiated recipients with 2 - 2.5 X 10(8) of the primary cultured cells and histologic EAE could be transferred with as few as 5 X 10(7) cells into naive recipients. This is the first demonstration of passive EAE induced with PLP-sensitized lymphoid cells and suggests the pathogenetic importance of cell-mediated immunity to PLP.     

Modulation of experimental autoimmune neuritis in Lewis rats by oral application of myelin antigens

Experimental autoimmune neuritis (EAN) in Lewis rats is a T cell-mediated disease and serves as an animal model of human inflammatory demyelinating neuropathies. EAN can be induced by immunization with complete bovine peripheral nerve myelin (BPM), the myelin protein P2 or its neuritogenic peptide, each emulsified in complete Freund's adjuvant (CFA). The present study evaluates the effect of oral tolerization with BPM or P2 protein on the development of actively induced EAN. Oral administration of BPM strongly suppressed clinical and histological signs of EAN subsequently induced by BPM/CFA, but feeding of P2 protein alone did not affect its course. In contrast, feeding of BPM did not mitigate the course of EAN subsequently induced by immunization with neuritogenic P2 peptide/CFA. Oral therapy with BPM after onset of myelin-induced EAN only slightly ameliorated the further course of disease, but significantly reduced lethality of this severe form of disease. The findings suggest that immunogenicity of the antigens fed determine strength of tolerance, that downregulation of EAN occurs at the site of immunization and not in the nerve, and that active suppression rather than specific anergization is operative in mediating resistance to EAN. However, only partial tolerance to myelin-induced EAN was achieved in naive animals by transfer of spleen/LN cells from rats orally tolerized with BPM. Although methodic factors may have limited the effect of the cells, the result is suggestive of some contribution of anergy to oral tolerance in the present model. Cholera toxin and LPS were identified as oral adjuvants for BPM and prolonged the state of tolerance. However, LPS exhibited proinflammatory properties if EAN was induced early after BPM/LPS-feeding. Thus, oral application of a mixture of myelin components in combination with cholera toxin may be a useful treatment for chronic inflammatory neuropathies considered autoimmune in nature.     

Attempts to suppress experimental allergic neuritis in the rat by pretreatment with antigen

Summary The injection of bovine dorsal root antigen in complete Freund's adjuvant can be used to produce experimental allergic neuritis (EAN) in rats. In this study attempts were made to prevent the development of the disease by prior injections of antigen. It was found that eight intradermal (i.d.) injections of antigen in either incomplete Freund's adjuvant or in saline failed to suppress EAN. A single intraperitoneal (i.p.) injection of antigen in saline produced only minimal protection against the disease. However, it was found that rats which had been given a primary course of EAN were subsequently completely unresponsive to a second injection of antigen.