Selenization of S. cerevisiae increases its protective potential in experimental autoimmune encephalomyelitis by triggering an intestinal immunomodulatory loop.

Multiple sclerosis is an autoimmune disease that affects the myelinated central nervous system (CNS) neurons and triggers physical and cognitive disabilities. Conventional therapy is based on disease-modifying drugs that control disease severity but can also be deleterious. Complementary medicines have been adopted and evidence indicates that yeast supplements can improve symptoms mainly by modulating the immune response. In this investigation, we evaluated the therapeutic potential of Saccharomyces cerevisiae and its selenized derivative (Selemax) in experimental autoimmune encephalomyelitis (EAE). Female C57BL/6 mice submitted to EAE induction were orally supplemented with these yeasts by gavage from day 0 to day 14 after EAE induction. Both supplements determined significant reduction in clinical signs concomitantly with diminished Th1 immune response in CNS, increased proportion of Foxp3+ lymphocytes in inguinal and mesenteric lymph nodes and increased microbiota diversity. However, Selemax was more effective clinically and immunologically; it reduced disease prevalence more sharply, increased the proportion of CD103+ dendritic cells expressing high levels of PD-L1 in mesenteric lymph nodes and reduced the intestinal inflammatory process more strongly than S. cerevisiae. These results suggest a clear gut-brain axis modulation by selenized S. cerevisiae and suggest their inclusion in clinical trials.

Blockade of sustained tumor necrosis factor in a transgenic model of progressive autoimmune encephalomyelitis limits oligodendrocyte apoptosis and promotes oligodendrocyte maturation.

BACKGROUND: Tumor necrosis factor (TNF) is associated with several neurodegenerative disorders including multiple sclerosis (MS). Although TNF-targeted therapies have been largely unsuccessful in MS, recent preclinical data suggests selective soluble TNF inhibition can promote remyelination. This has renewed interest in regulation of TNF signaling in demyelinating disease, especially given the limited treatment options for progressive MS. Using a mouse model of progressive MS, this study evaluates the effects of sustained TNF on oligodendrocyte (OLG) apoptosis and OLG precursor cell (OPC) differentiation. METHODS: Induction of experimental autoimmune encephalomyelitis (EAE) in transgenic mice expressing a dominant-negative interferon-γ receptor under the human glial fibrillary acidic protein promoter (GFAPγR1Δ) causes severe non-remitting disease associated with sustained TNF. Therapeutic effects in GFAPγR1Δ mice treated with anti-TNF compared to control antibody during acute EAE were evaluated by assessing demyelinating lesion size, remyelination, OLG apoptosis, and OPC differentiation. RESULTS: More severe and enlarged demyelinating lesions in GFAPγR1Δ compared to wild-type (WT) mice were associated with increased OLG apoptosis and reduced differentiated CC1+Olig2+ OLG within lesions, as well as impaired upregulation of TNF receptor-2, suggesting impaired OPC differentiation. TNF blockade during acute EAE in GFAPγR1Δ both limited OLG apoptosis and enhanced OPC differentiation consistent with reduced lesion size and clinical recovery. TNF neutralization further limited increasing endothelin-1 (ET-1) expression in astrocytes and myeloid cells noted in lesions during disease progression in GFAPγR1Δ mice, supporting inhibitory effects of ET-1 on OPC maturation. CONCLUSION: Our data implicate that IFNγ signaling to astrocytes is essential to limit a detrimental positive feedback loop of TNF and ET-1 production, which increases OLG apoptosis and impairs OPC differentiation. Interference of this cycle by TNF blockade promotes repair independent of TNFR2 and supports selective TNF targeting to mitigate progressive forms of MS.

Voluntary wheel running differentially affects disease outcomes in male and female mice with experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an inflammatory neurodegenerative disease of the central nervous system. The primary symptoms of MS include the loss of sensory and motor function. Exercise has been shown to modulate disease parameters in experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, by reducing immune cell infiltration and oxidative stress. However, these initial studies were carried out exclusively in female mice. The present study compared the effects of daily voluntary wheel running on several disease parameters in male and female mice with EAE. Male and female mice were given access to a running wheel for 1h a day for 30 consecutive days. Daily wheel running significantly improved clinical scores in males with EAE but had little effect on clinical signs in females with the disease. Direct comparison of inflammation, axonal injury, and oxidative stress in male and female mice with EAE revealed significant differences in the amount of T-cell infiltration, microglia reactivity, demyelination and axon integrity. Male mice with EAE given daily access to running wheels also had significantly less ongoing oxidative stress compared to all other groups. Taken together, our results indicate that the inflammatory response generated in EAE is distinct between the sexes and its modulation by daily exercise can have sex-specific effects on disease-related outcomes.

Dietary cholesterol promotes repair of demyelinated lesions in the adult brain.

Multiple Sclerosis (MS) is an inflammatory demyelinating disorder in which remyelination failure contributes to persistent disability. Cholesterol is rate-limiting for myelin biogenesis in the developing CNS; however, whether cholesterol insufficiency contributes to remyelination failure in MS, is unclear. Here, we show the relationship between cholesterol, myelination and neurological parameters in mouse models of demyelination and remyelination. In the cuprizone model, acute disease reduces serum cholesterol levels that can be restored by dietary cholesterol. Concomitant with blood-brain barrier impairment, supplemented cholesterol directly supports oligodendrocyte precursor proliferation and differentiation, and restores the balance of growth factors, creating a permissive environment for repair. This leads to attenuated axon damage, enhanced remyelination and improved motor learning. Remarkably, in experimental autoimmune encephalomyelitis, cholesterol supplementation does not exacerbate disease expression. These findings emphasize the safety of dietary cholesterol in inflammatory diseases and point to a previously unrecognized role of cholesterol in promoting repair after demyelinating episodes.

Thalamus Degeneration and Inflammation in Two Distinct Multiple Sclerosis Animal Models.

There is a broad consensus that multiple sclerosis (MS) represents more than an inflammatory disease: it harbors several characteristic aspects of a classical neurodegenerative disorder, i.e., damage to axons, synapses, and nerve cell bodies. While several accepted paraclinical methods exist to monitor the inflammatory-driven aspects of the disease, techniques to monitor progression of early and late neurodegeneration are still in their infancy and have not been convincingly validated. It was speculated that the thalamus with its multiple reciprocal connections is sensitive to inflammatory processes occurring in different brain regions, thus acting as a "barometer" for diffuse brain parenchymal damage in MS. To what extent the thalamus is affected in commonly applied MS animal models is, however, not known. In this article we describe direct and indirect damage to the thalamus in two distinct MS animal models. In the cuprizone model, we observed primary oligodendrocyte stress which is followed by demyelination, microglia/astrocyte activation, and acute axonal damage. These degenerative cuprizone-induced lesions were found to be more severe in the lateral compared to the medial part of the thalamus. In MOG35-55-induced EAE, in contrast, most parts of the forebrain, including the thalamus were not directly involved in the autoimmune attack. However, important thalamic afferent fiber tracts, such as the spinothalamic tract were inflamed and demyelinated on the spinal cord level. Quantitative immunohistochemistry revealed that this spinal cord inflammatory-demyelination is associated with neuronal loss within the target region of the spinothalamic tract, namely the sensory ventral posterolateral nucleus of the thalamus. This study highlights the possibility of trans-neuronal degeneration as one mechanism of secondary neuronal damage in MS. Further studies are now warranted to investigate involved cell types and cellular mechanisms.

Thrombin mutant W215A/E217A treatment improves neurological outcome and attenuates central nervous system damage in experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is a neuroinflammatory disease characterized by demyelination and axonal damage of the central nervous system. The pathogenesis of MS has also been linked to vascular inflammation and local activation of the coagulation system, resulting in perivascular fibrin deposition. Treatment of experimental autoimmune encephalomyelitis (EAE), a model of human MS, with antithrombotic and antiinflammatory activated protein C (APC) reduces disease severity. Since recombinant APC (Drotecogin alfa), originally approved for the treatment of severe sepsis, is not available for human MS studies, we tested the hypothesis that pharmacologic activation of endogenous protein C could likewise improve the outcome of EAE. Mice were immunized with murine myelin oligodendrocyte glycoprotein (MOG) peptides and at the onset of EAE symptoms, were treated every other day with either WE thrombin (25 µg/kg; i.v.), a selective recombinant protein C activator thrombin analog, or saline control. Mice were monitored for changes in disease score until euthanized for ex vivo analysis of inflammation. Administration of WE thrombin significantly ameliorated clinical severity of EAE, reduced inflammatory cell infiltration and demyelination, suppressed the activation of macrophages comprising the CD11b + population and reduced accumulation of fibrin (ogen) in the spinal cord. These data suggest that symptomatic MS may respond to a treatment strategy that involves temporal pharmacological enhancement of endogenous APC generation.

Effect of aqueous extract of Achillea millefolium on the development of experimental autoimmune encephalomyelitis in C57BL/6 mice.

OBJECTIVE: Achillea millefolium (A. millefolium) is widely used as an anti-inflammatory remedy in traditional and herbal medicine. In this study, we investigated the effect of an aqueous extract from A. millefolium on experimental autoimmune encephalomyelitis (EAE) and on the serum cytokine levels in C57BL/6 mice. MATERIALS AND METHODS: EAE was induced in 63 C57BL/6 mice weighing 20-25 g (8 weeks old). Following immunization, the treatment protocol was initiated by using different doses of an aqueous extract from A. millefolium (1, 5, and 10 mg/mouse/day). Histopathologic assessments were performed by hematoxylin and eosin (H and E) and luxol fast blue (LFB) staining. Behavioral disabilities were recorded by a camera. Serum levels of interleukin (IL)-10, IL-12, and transforming growth factor (TGF)-ß were measured using enzyme-linked immunosorbent assay (ELISA). RESULTS: On average, mice developed classical behavioral disabilities of EAE, 13.2 ± 1.9 days following immunization. Treatment of mice with A. millefolium led to delay the appearance of behavioral disabilities along with reduced severity of the behavioral disabilities. Treatment with A. millefolium prevented weight loss and increased serum levels of TGF-ß in immunized mice with MOG35-55. EAE-induced mice, which were treated with A. millefolium, had less cerebral infiltration of inflammatory cells. CONCLUSION: The results demonstrated that treatment with aqueous extract of A. millefolium may attenuate disease severity, inflammatory responses, and demyelinating lesions in EAE-induced mice. In addition, following treatment with A. millefolium, serum levels of TGF-ßwere increased in EAE-induced mice.

Metallothioneins I/II expression in rat strains with genetically different susceptibility to experimental autoimmune encephalomyelitis.

OBJECTIVES: Compared to the Dark Agouti (DA), the Albino Oxford (AO) rat strain exhibits lower susceptibility to the induction of experimental autoimmune encephalomyelitis (EAE). Here, we investigated the potential contribution of the heavy metal-binding proteins metallothioneins (MTs) I/II to these effects. METHODS: Rats were immunized with bovine brain homogenate emulsified in complete Freund's adjuvant or only with complete Freund's adjuvant. The expression patterns of MTs mRNA and proteins and tissue concentrations of Zn2+ and Cu2+ were estimated in the brain and in the liver on days 7 and 12 after immunization, by real-time PCR, immunohistochemistry and inductively coupled plasma spectrometry, respectively. Additionally, the hepatic transforming growth factor beta and nuclear factor kappa B immunoreactivities were tested. RESULTS: Clinical signs of EAE were not induced in AO rats, but they upregulated the expression of MT I/II proteins in the brain (hippocampus and cerebellum) and in the liver, similarly as DA rats. The transcriptional activation of MT-I occurred, however, only in DA rats, which accumulated also more zinc in the brain and in the liver. In contrast, intact AO rats had greater hepatic MT-I mRNA immunoreactivity and more Cu2+ in the hippocampus. Besides, in immunized AO rats a high upregulation of transforming growth factor beta and nuclear factor kappa B immunoreactivities was found in several hepatic structures (vascular endothelium, Kupffer cells and hepatocytes). CONCLUSIONS: Our data show that AO and DA rats differ in constitutive and inductive MT-I gene expression in the brain and in the liver, as well as in the hepatic cytokine profile, suggesting that these mechanisms may contribute to the discrepancy in the susceptibility to EAE.

Assessment of evidence for a protective role of vitamin D in multiple sclerosis.

Evidence for a role of vitamin D insufficiency in determining risk in Multiple Sclerosis (MS) is supported by studies in both pediatric- and adult-onset patients. The potential role of vitamin D in modulating MS disease activity is an area of active clinical trials research, and the possibility of primary disease prevention with vitamin D supplementation in early life is an emerging concept. With Sir Austin Bradford Hill's criteria as a framework, the present review assesses the evidence for a causal relationship between vitamin D insufficiency and the pathobiology of MS, and discusses rationale for future clinical trials with vitamin D.

A Lecinoxoid, an oxidized phospholipid small molecule, constrains CNS autoimmune disease.

Oxidized phospholipids (Ox-PLs) are generated in abundance at sites of inflammation. Recent studies have indicated that Ox-PLs may also exhibit anti-inflammatory activities. In this study, we investigated the beneficial effect of VB-201, a pure synthetic Ox-PL analog that we synthesized, on the development of a central nervous system (CNS) autoimmune inflammatory disease, in vivo. Oral administration of VB-201 ameliorated the severity of experimental autoimmune encephalomyelitis (EAE) induced by myelin oligodendrocyte glycoprotein (MOG) peptide MOG35-55, and restrained the encephalogenicity of MOG35-55-specific T-cells. Our data presents a novel prospect for the role of Ox-PL analogs in CNS inflammatory diseases.

Vitamin D3 confers protection from autoimmune encephalomyelitis only in female mice.

The prevalence of multiple sclerosis (MS) increases significantly with decreasing UV B light exposure, possibly reflecting a protective effect of vitamin D(3). Consistent with this theory, previous research has shown a strong protective effect 1,25-dihydroxyvitamin D(3) in experimental autoimmune encephalomyelitis (EAE), an MS model. However, it is not known whether the hormone precursor, vitamin D(3), has protective effects in EAE. To address this question, B10.PL mice were fed a diet with or without vitamin D(3), immunized with myelin basic protein, and studied for signs of EAE and for metabolites and transcripts of the vitamin D(3) endocrine system. The intact, vitamin D(3)-fed female mice had significantly less clinical, histopathological, and immunological signs of EAE than ovariectomized females or intact or castrated males. Correlating with reduced EAE, the intact, vitamin D(3)-fed female mice had significantly more 1,25-dihydroxyvitamin D(3) and fewer CYP24A1 transcripts, encoding the 1,25-dihydroxyvitamin D(3)-inactivating enzyme, in the spinal cord than the other groups of mice. Thus, there was an unexpected synergy between vitamin D(3) and ovarian tissue with regard to EAE inhibition. We hypothesize that an ovarian hormone inhibited CYP24A1 gene expression in the spinal cord, so the locally-produced 1,25-dihydroxyvitamin D(3) accumulated and resolved the inflammation before severe EAE developed. If humans have a similar gender difference in vitamin D(3) metabolism in the CNS, then sunlight deprivation would increase the MS risk more significantly in women than in men, which may contribute to the unexplained higher MS incidence in women than in men.

Nitric oxide and the immunomodulation of experimental allergic encephalomyelitis.

Previous studies examining the effect of nitric oxide synthase (NOS) inhibition on the course of experimental allergic encephalomyelitis (EAE) have yielded conflicting results. This may relate to the use of nonspecific inhibitors and to differences between active and adoptive EAE. We examined the effect of treatment with L-N-(1-iminoethyl)lysine (L-NIL), a selective inhibitor of the cytokine-inducible isoform of NOS, on the clinical course of active and adoptive EAE in Lewis rats. We find that while L-NIL treatment of recipients is protective in adoptive EAE, treatment of active EAE with L-NIL leads to a marked accentuation of disease expression. In L-NIL-treated animals treated with myelin basic protein/complete Freund's adjuvant (MBP/CFA), disease onset is accelerated and clinical symptoms are more severe. Accentuation of integrated disease scores is seen even if L-NIL treatment is started 5 days following immunization. The histological findings in involved spinal cords from L-NIL-treated animals with active EAE are similar to those from untreated animals with similar clinical scores. L-NIL treatment of MBP/CFA-immunized animals does not prevent recovery from clinical symptoms, nor does it allow for reinduction of disease in animals previously immunized with MBP/CFA. Treatment of F344 rats, a strain which is relatively nonsusceptible for EAE, with L-NIL results in consistent evidence of EAE following immunization with MBP/CFA. These findings, together with our previous work on interstitial nephritis, support a role for endogenously generated NO in immunoregulation of T cell responses following immunization with antigen in CFA, and suggest that inducibility of NOS expression may be an important susceptibility factor for autoimmunity.

Melanin, melatonin, melanocyte-stimulating hormone, and the susceptibility to autoimmune demyelination: a rationale for light therapy in multiple sclerosis.

Multiple sclerosis is a central nervous system demyelinating disease. Significant evidence, including similarities with its animal model, experimental autoimmune encephalomyelitis, supports an autoimmune mechanism, activated by putative environmental factors in genetically predisposed individuals. Genetic factors strongly influence the susceptibility to demyelinating diseases in humans and rodents. Understanding the mechanisms governing susceptibility versus resistance may help to identify individuals at risk or design therapeutic strategies. The hypothesis formulated here is based on the observation that resistance to multiple sclerosis and experimental autoimmune encephalomyelitis is associated with dark skin pigmentation. While this may signify a protective role for melanin against environmental factors producing oxidative damage, the mechanism postulated here is that susceptibility to autoimmune demyelination is influenced by hormonal factors, i.e. the neurohormones melatonin and melanocyte stimulating hormone, which have opposing effects on immune functions and, the same time, are important determinants of the individual's production of melanin.

Reactive oxygen species are involved in the pathogenesis of experimental allergic encephalomyelitis in Lewis rats.

During experimental allergic encephalomyelitis (EAE), both blood-borne macrophages as well as activated, resident microglial cells are considered to be involved in inflammatory reactions in the central nervous system (CNS), resulting in the neurological deficits common to EAE. Both cell types can produce multiple mediators of tissue damage, among which are the reactive oxygen species (ROS). In this study we show that macrophages and microglial cells, isolated from the CNS of Lewis rats with clinical signs of EAE, exhibited significantly elevated spontaneous and phorbol myristate acetate (PMA)-inducible levels of ROS compared to similar cells isolated from healthy controls, sham (complete Freund's adjuvant, CFA)-immunized rats as well as rats sacrificed before the manifestation of clinical signs of EAE. However, during clinical EAE, peripheral blood mononuclear cells (PBMC) did not show increased spontaneous nor PMA-inducible ROS production compared to controls. In vivo treatment of EAE with catalase, which scavenges the ROS H2O2, markedly suppressed the severity of the disease as compared to sham (albumin)-treated controls. In contrast, superoxide dismutase had no effect on clinical signs. Our studies point at a putative functional role for ROS, and in particular H2O2, in the pathogenesis of EAE.

[Cell therapy and its risks]. / Zelltherapie und ihre Gefahren.

Parenteral administration of fresh cells, frozen cells (snapfrozen cell suspensions) or lyophilized cells (sicca cells), is known as cellular or cell therapy. While the German Health Office (BGA) provisionally banned the use of dried cell preparations in 1987, injection of fresh cells is still allowed. There have been repeated reports of life-threatening, and even fatal, complications of this type of therapy. Since it involves the administration of heterologous biological material, most of the complications that have been observed have been of the allergic/hyperergic type resembling experimental allergic encephalomyelitis (EAE) and experimental allergic neuritis (EAN). When evaluating the risks of this form of alternative-medical treatment, the well-known risks of injection therapy must also be borne in mind. In the case of cell therapy, too, the hoped for effect must be weighed against the risks of the procedure, and our guiding principle must be: nil nocere.

Disruption of the blood-brain barrier in experimental optic neuritis: immunocytochemical co-localization of H2O2 and extravasated serum albumin.

PURPOSE: To probe the role of endogenous hydrogen peroxide (H2O2) in the pathogenesis of disruption of the blood-brain barrier (BBB) associated with experimental allergic encephalomyelitis (EAE), an animal model for primary central nervous system demyelination. METHODS: Strain-13 guinea pigs were sensitized for EAE with central myelin in complete Freund's adjuvant. Magnetic resonance imaging with Gd-DTPA was performed twice a week for 2 weeks to assess disruption of the BBB, in vivo, by the enhancement of the optic nerves. Two weeks after antigenic sensitization, ultracytochemical localization of endogenous H2O2 was performed using the cerium perhydroxide method, with co-localization of endogenous serum albumin extravasation using gold-labeled antibodies against serum albumin. Examination of blood vessels for perivascular immunogold-labeled serum albumin and H2O2 derived reaction product began in the optic nerve head and proceeded toward the retrobulbar optic nerve until a total of 20 vessels were evaluated per animal. RESULTS: Magnetic resonance imaging revealed Gd-DTPA enhancement of the optic nerves in all animals sensitized for EAE. Optic nerve ultrastructure revealed colloidal gold-labeled antibodies against serum albumin in the perivascular and adjacent interstitial spaces of capillaries and small venules in which H2O2 derived cerium perhydroxide reaction product was also simultaneously evident. Immunogold-labeled serum albumin was predominantly confined to the intravascular compartment of the optic nerve in the absence of perivascular H2O2 and/or perivascular foci of inflammatory cells. The difference between the mean percentage of blood vessels (61.8%) with co-localization of perivascular immunogold-labeled serum albumin and cerium perhydroxide reaction product, to the mean percentage of blood vessels (9.5%) with perivascular immunogold-labeled serum albumin in the absence of cerium perhydroxide, was statistically significant (P = 0.0019). CONCLUSIONS: Endogenous H2O2, found at the foci of BBB disruption, may be one of the mediators involved in the alteration of vascular permeability in experimental optic neuritis.

Delta 9-tetrahydrocannabinol: a novel treatment for experimental autoimmune encephalomyelitis.

Since multiple sclerosis (MS) is believed to be an immune-mediated disease, it follows that its therapies should be directed towards modulating the immune system. Current MS treatments, which include the use of exogenous steroids that are immunosuppressive, do not meet therapeutic objectives. delta 9-Tetrahydrocannabinol (THC), an active component of marijuana, has been shown to be immunosuppressive. To test THC's ability to suppress an immune-mediated disease, experimental autoimmune encephalomyelitis (EAE), the laboratory model of MS, was used. Lewis rats and strain 13 guinea pigs were administered THC either before inoculation for EAE or treated with THC after injection. Control animals received placebo. The effect of dose, in addition to the timing of treatment, was also investigated. All animals treated with placebo developed severe clinical EAE 10-12 days post-injection (d.p.i.) and more than 98% died by 15 d.p.i. THC-treated animals had either no clinical signs or mild signs with delayed onset (13-15 d.p.i.) with survival greater than 95%. Examination of central nervous system tissue revealed a marked reduction of inflammation in the THC-treated animals. Therefore, as THC has been shown to inhibit both clinical and histologic EAE, it may prove to be a new and relatively innocuous agent for the treatment of immune-mediated diseases.

The role of antigen absorption in the resistance of brown-Norway rats to experimental allergic encephalomyelitis.

The designation of Brown-Norway (BN) rats as resistant to experimental allergic encephalomyelitis (EAE) is an oversimplification. Lewis rats are susceptible and BN rats are usually resistant to EAE after inoculation with guinea pig spinal cord or basic protein in Freund's adjuvant. However, EAE can be produced in BN rats by immunizing with rat cord and carbonyl iron, a particulate adjuvant. In the present work, the possibility that susceptibility of BN rats under these conditions is due only to the special qualities of the adjuvant has been eliminated by producing EAE in them without any adjuvant at all, merely by increasing the absorption and processing of the rat cord antigen. The susceptibility of the F1 hybrids is intermediate with respect to guinea pig cord antigen but it was equal to or greater than either parental strain when tested with rat cord antigen. Histologic evidence that BN rats do not absorb or process neural antigen as well as other strains, and the augmentation of EAE by increasing the dose and absorption of the inoculum, suggest that antigen absorption, processing and presentation is a "bottleneck" for development of EAE in BN rats. Absorption and processing of antigen should be considered along with cellular response, inflammatory mediators and epitope dominance when analyzing susceptibility and resistance to EAE.

Serum anti-myelin antibodies in chronic relapsing experimental allergic encephalomyelitis.

To investigate the role of anti-myelin antibodies in chronic relapsing experimental allergic encephalomyelitis (CR-EAE), sera from SJL/J mice with CR-EAE actively induced by inoculation with spinal cord homogenate in complete Freund's adjuvant (CFA) were compared with sera from mice to whom CR-EAE was passively transferred by lymph node cells (LNC) stimulated with myelin basic protein (BP). Sera were obtained serially from mice during both remissions and relapses of disease and were evaluated for the presence of anti-myelin antibodies using an avidin-biotin-immunoperoxidase technique. Four of six mice with CR-EAE induced with cord-CFA were positive for anti-myelin antibodies 15-124 days after inoculation, with 16 of 18 sera positive in these four mice. Two mice inoculated with cord-CFA did not have detectable serum anti-myelin antibodies, despite a clinical and histopathological picture indistinguishable from the antibody-positive mice. None of seven mice with CR-EAE passively transferred by BP-stimulated LNC had detectable anti-myelin antibodies in 30 sera obtained 7-141 days after cell transfer. We conclude that serum anti-myelin antibodies probably do not play a significant role in the pathogenesis of CR-EAE in SJL/J mice.