International Immunology

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International Immunology, Vol. 13, No. 3, 257-264, March 2001
© 2001 Japanese Society for Immunology

Identification of genetic loci associated with paralysis, inflammation and weight loss in mouse experimental autoimmune encephalomyelitis

Jeffrey A. Encinas¹, Marjorie B. Lees^2,3, Raymond A. Sobel⁴, Cammie Symonowicz³, Howard L. Weiner¹, Christine E. Seidman⁵, J. G. Seidman⁵ and Vijay K. Kuchroo¹

¹ Center for Neurologic Diseases, Department of Medicine, Brigham & Women's Hospital and
² Department of Neurology, Harvard Medical School, Boston, MA 02115, USA
³ E. K. Shriver Center, Waltham, MA 02254, USA
⁴ Department of Pathology, Stanford University School of Medicine and Laboratory Service, Veterans Health Care System, Palo Alto, CA 94304, USA
⁵ Department of Genetics, Howard Hughes Medical Institute, Harvard Medical School, Boston, MA 02115, USA

Correspondence to: V. K. Kuchroo

	Abstract

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Experimental autoimmune encephalomyelits (EAE), a model for human multiple sclerosis, is an inducible inflammatory and demyelinating disease of the central nervous system (CNS). Susceptibility to this disease is heritable and is demonstrated by the development of an ascending paralysis accompanied by a loss in body wt 2–3 weeks following immunization with proteins derived from CNS myelin. In a previous genetic analysis of susceptibility to EAE in a cross between susceptible SJL/J mice and resistant B10.S mice, we found suggestive evidence of linkage with disease susceptibility at the telomeric end of chromosome 2 and in the central region of chromosome 3. To define these associations more precisely and to investigate the genetic factors controlling measurable phenotypes of EAE, we performed a new analysis with a larger number of mice. The results now indicate that the chromosome 2 locus significantly influences EAE-related weight loss (P = 6.7 x 10^–5) and that the chromosome 3 locus is linked with the development of paralysis. In addition, an intriguing inheritance pattern was revealed in which female backcross mice generated from B10.S female x (B10.S x SJL/J)F₁ male parents experienced significantly more EAE-related weight loss (P = 1.2 x 10^–4) than females generated from F₁ female x B10.S male parents. After controlling for this inheritance, a new locus at the centromeric end of chromosome 8 was identified that significantly influences both the development of paralysis (P = 8.2 x 10^–6) and the incidence of CNS inflammation (P = 7.0 x 10^–5) in EAE.

Keywords: C57BL, chromosome mapping, disease susceptibility, genetic crosses, genetic markers, human, inbred strains, incidence, linkage, multiple sclerosis, SJL, quantitative trait

	Introduction

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Experimental autoimmune encephalomyelitis (EAE) is a commonly studied autoimmune disease because of its relatively rapid induction time and because its major clinical manifestation, paralysis, can be assessed easily by a simple visual inspection. The disease is induced by immunization with myelin proteins and is known to be mediated by T cells that traffic to the central nervous system (CNS), and initiate events that lead to tissue injury and clinical signs. Its development, severity and time course are influenced by complex genetic and environmental factors that are incompletely understood (1,2).

In a previous genetic linkage analysis of susceptibility to EAE using a cross between susceptible SJL/J mice and resistant B10.S mice, we found no genetic loci from the SJL/J genome that were absolutely required for susceptibility (3). We did, however, identify several loci that showed evidence of linkage with susceptibility. In that study, the genotypes of 68 highly susceptible backcross mice were compared with the genotypes of 68 disease-resistant backcross mice by carrying out a genome scan using 130 microsatellite markers covering all chromosomes. For the linkage analysis, the trait of disease susceptibility was treated as a qualitative trait where affected mice were considered susceptible and unaffected mice were considered not susceptible. As a result, a locus on chromosome 2 and another on chromosome 3 were found that associated with susceptibility at P = 0.001. Eight other regions were found with weak associations of P < 0.05 on chromosomes 4, 6, 8 (centromeric), 8 (medial), 9, 15, 16 and 18. This finding of a complex inheritance pattern is in agreement with genetic linkage results reported by others (4–7) and suggests that EAE susceptibility is a polygenic trait in which small differences in the expression or activity of different gene products combine to determine the likelihood of disease.

To study the genetic factors controlling susceptibility to EAE further, therefore, we performed a new linkage analysis using different disease phenotypes of EAE and increased the number of mice used in the analysis. The power of the genetic analysis was increased by examining so-called intermediate phenotypes of the disease, which may be more influenced by a small subset of the disease-associated loci, and attempting to apply a quantitative trait locus analysis wherever possible. Mice with varying degrees of susceptibility were phenotyped according to the severity of their clinical signs and genotyped at all chromosomal locations where associations with disease susceptibility were found in the initial analysis. This has allowed us to find significant linkages with disease and has enabled us to determine approximately the traits affected by each locus.

For this analysis, 298 (SJL/JxB10.S)F₁xB10.S backcross mice varying in disease severity from mild or no clinical disease to severe paralysis of all limbs were used. Severity of disease was measured by noting the day of onset, scoring the extent of paralysis, measuring changes in body wt and assessing the extent of CNS inflammation. Genotyping of the chromosomes that were suggested from the preliminary analysis as likely to be carrying genes affecting EAE susceptibility was done using microsatellite markers.

Whereas we previously showed suggestive evidence that loci on chromosomes 2 and 3 were linked with susceptibility to EAE, we now show that the chromosome 2 locus significantly influences EAE-related weight loss and that the chromosome 3 locus influences the development of paralysis. Additionally, we identified an interesting pattern of inheritance in which female backcross mice showed a very different response to the immunization depending on whether their F₁ parent was male or female. Once this was recognized, analysis was done on subsets of mice to take advantage of this difference, and a new locus that was linked significantly with the development of paralysis and the extent of inflammation in the CNS was uncovered at the centromeric end of chromosome 8.

	Methods

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Mice
Parental B10.S and SJL/J breeding pairs were obtained from the Jackson Laboratory (Bar Harbor, ME) and were kept at the E. K. Shriver Center (Waltham, MA). (B10.SxSJL/J) F₁ and (F₁xB10.S) backcross mice were bred in this facility, and all mice were maintained under identical conditions. F₁ and backcross breeding was set up without preference for whether the B10.S strain was used as the father or the mother in the mating.

Phenotyping
To induce EAE, mice were immunized at 8–12 weeks of age by injecting s.c. into the flanks 0.2 ml of an emulsion made by mixing equal volumes of 1 mg/ml proteolipid protein (PLP) peptide 139–151 (HSLGKWLGHPDKF) in PBS and 4 mg/ml Mycobacteria tuberculosis H37Ra (Difco, Detroit, MI) in complete Freund's adjuvant (Difco). Each mouse was also given an injection of 4.5x10⁹ Bordetella pertussis bacilli (pertussis vaccine; Massachusetts Public Health Biologics Laboratories, Boston, MA) into the tail vein on the day of immunization and 2 days later. The PLP peptide was synthesized in the laboratory of Dr Richard Laursen (Department of Chemistry, Boston University, Boston, MA) using Fmoc chemistry. Mice were observed regularly for signs of disease for up to 4 weeks. To assure adequate access to food and water, moistened chow pellets were provided at cage floor level and fed by hand to severely paralyzed mice. Severity of paralysis was measured on a scale of 0 to 5 where 0 = no paralysis, 1 = limp tail, 2 = limp tail and weak gait, 3 = hind limb paralysis, 4 = fore and hind limb paralysis, and 5 = moribund. Weights of mice were measured approximately every other day beginning on day 7 after immunization. Weight loss was calculated as [(day 7 weight – final weight)/day 7 weight]x100%, where final weight is the weight of the mouse on the day of sacrifice.

Mice were sacrificed when they exhibited a clinical score of 4 or when they began to recover from disease as indicated by weight gain and no further increase in clinical score. Mice that did not develop paralysis were sacrificed between day 28 and 30 after immunization. Brains and spinal cords from the mice were taken for histological examination, and livers were taken as a source of DNA for genotyping. Mice that died spontaneously during the study were excluded from the analysis because of the poor tissue quality for the histological evaluation and the uncertainty that death was due to EAE. Brains and spinal cords were fixed in 10% phosphate-buffered formalin and paraffin-embedded sections were stained with luxol fast blue–hematoxylin & eosin for light microscopy. Histological disease was quantitated by counting the inflammatory foci in meninges and parenchyma as previously described (8).

Genotyping
Livers were taken from all treated mice and frozen at –70°C. DNA was prepared by digesting homogenized tissue with proteinase K and performing a phenol–chloroform extraction according to standard protocols (9). Microsatellite marker loci were chosen at ~15 cM intervals based on genetic maps of the mouse (10–12) and PCR primers (MapPairs, Research Genetics, Huntsville, AL) for these were purchased. PCR amplification was performed either in a Perkin-Elmer Cetus (Norwalk, CT) 9600 thermal cycler or a PTC-100 model thermal cycler (MJ Research, Watertown, MA) in 20 µl volumes using 100 ng genomic DNA, 0.2 mM dNTPs (Boeringer Mannheim, Mannheim, Germany), 150 nM of each primer and 1.0 U of Taq DNA polymerase, in standard PCR buffer containing 1.5 mM MgCl₂. Forty cycles of 94°C for 20 s, 55°C for 30 s and 72°C for 30 s was generally used, although some primer pairs required a slightly higher or lower annealing temperature for optimum amplification. All PCR products were electrophoresed on a 3% MetaPhor (FMC Bioproducts, Rockland, ME) agarose gel and stained with ethidium bromide to test for polymorphisms between the parental strains. The majority of polymorphic markers had size differences discernible on the 3% MetaPhor agarose gel. For those with smaller size differences, PCR amplification was performed by substituting forward primers end-labeled with [³²P]ATP using T4 polynucleotide kinase (Boeringer Mannheim) and the products were electrophoresed on 6% denaturing polyacrylamide gels. The gels were exposed to films for 6–12 h at room temperature for visualization.

Backcross animals were scored at each locus as either homozygous or heterozygous by comparing the sizes of their amplified products with simultaneously amplified products from each of the parental inbred strains.

Statistics
Linkage analysis was carried out using the computer program Map Manager QT (http://mcbio.med.buffalo.edu/mmQT.html) which uses linear regression to interval map quantitative trait loci. Output from this program is in the form of a linkage ratio statistic (LRS) which follows a ² distribution and can be easily converted to a P value. The LRS can be converted to a Lod score by dividing by 2ln₁₀. Following the guidelines proposed by Lander and Kruglyak, P < 1.0x10^–4 (Lod score 3.3) was considered to be the cut-off for significant linkage and P < 3.4x10^–3 (Lod score 1.9) was considered to be suggestive of linkage (13). Student's t-test was used to evaluate the differences in the means of quantitative traits between sets of mice. Corrections for multiple comparisons were made using the Bonferroni method. Normality was determined by a normal quantile plot.

	Results

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Paralysis, day of onset, inflammation and weight loss as EAE phenotypes
Quantitative trait analyses assume that multiple alleles contribute to a trait so that with random assortment of the alleles, different magnitudes of the trait will follow a normal distribution in a population (14). These different trait magnitudes can then be correlated with genotype to determine linkage of loci with the trait. Among the EAE-related traits that we measured, weight loss was a good quantitative trait, whereas paralysis, day of onset and inflammation had limitations associated with their assessment that affected their ability to be used as normal quantitative traits.

The first trait, paralysis, is a result of decreased or slowed transmission of nerve signals through myelinated nerves that may be caused either by destruction of myelin and denuding of axons (15) or by edema that disrupts the function of axonal nodes of Ranvier (16). In the mice used in these experiments, paralysis characteristically first developed in the tail then progressed to affect the hind limbs and finally the fore limbs, suggesting that the extent of paralysis reflects the magnitude, rather than the location, of the inflammatory response in the CNS. Scoring of paralysis ranges from 0, where no paralysis is evident, to 4, where paralysis has progressed to involve the forelimbs. This scoring system differentiates between mice with different degrees of paralysis, but cannot differentiate between mice that experience varying milder degrees of myelin destruction or edema but do not reach a threshold of functional disruption that manifests in paralysis. All of these latter mice are scored as 0 and as a result the curve for the distribution of paralytic scores is truncated at the threshold and is not normal. A similar truncation occurs at the upper end of the scale. Because of this, our analysis was carried out with paralysis scored qualitatively (0 = no paralysis, 1 = paralysis), i.e. the incidence of paralysis, rather than its severity, was used in the analysis.

The second trait, early onset of paralytic disease, may indicate a low threshold for activation of an immune response directed at self, a lack of immune regulatory mechanisms, a weak barrier to inflammatory cells entering the CNS or a heightened sensitivity to immune-mediated damage in the CNS. Similar to the measurement of paralysis severity, however, the day of paralysis onset can only be measured among affected mice. Therefore, day of onset was analyzed as a quantitative trait, but mice that did not show signs of paralysis were excluded from the analysis.

The third trait, inflammation in the CNS after immunization, is an indication of successful immune activation. Susceptible mice, however, typically experience an initial episode of clinical disease followed by remissions and relapses that occur at unpredictable intervals, and inflammation in the CNS waxes and wanes along with the disease (17). In addition, changes in the severity of paralysis lag behind changes in the number of histologically observable lesions by an unpredictable amount of time (18). By measuring the number of inflammatory foci at only a single time point, foci number may have decreased substantially from the maximum in many cases if mice had already begun to recover when their CNS tissue was taken and therefore may not be representative of the true magnitude of inflammation that had occurred. For this reason, the presence or absence, rather than the extent, of inflammation in the CNS was used in the analysis.

The fourth trait, severe weight loss, is a common feature of EAE that usually accompanies paralysis. The weight loss is most likely due to side effects of inflammatory mediators (19–21), but can also result from a suppression of appetite or ability to adequately hydrate. After immunization, mice were weighed regularly, making it possible to approximate the maximum weight loss induced by disease. Since mice that are susceptible to disease begin losing weight 8–10 days after immunization, we took an initial weight on day 7, then weighed the mice every other day thereafter until day 28. Mice that developed paralysis were weighed until they either developed a clinical score of 4 or showed signs of recovery from disease. To adjust for differences in initial body wt, weight loss was calculated as the percentage change in body wt from the weight taken on day 7. Amounts of weight loss among the 298 mice followed a normal continuous distribution as shown in Fig. 1. Therefore, an analysis of weight change as a quantitative trait was performed on the mice.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Distribution of weight change among all backcross mice. Bars represent the number of mice that changed their weight by an amount that falls into one of the 5% intervals indicated on the x-axis.

 
Linkage analysis
Linkage analysis was carried out using 298 backcross mice for the traits of paralysis, inflammation, weight loss and day of paralysis onset. One locus at the telomeric end of chromosome 2 (at microsatellite marker D2Mit200), near the locus at which we had previously found suggestive linkage with susceptibility, showed significant linkage with weight loss at P = 6.7x10^–5 (Fig. 2). Suggestive linkage with weight loss was seen near D8Mit84 on chromosome 8 (P = 1.4x10^–3). Suggestive linkage was also found with paralysis near D8Mit226 on chromosome 8 (P = 4.3x10^–4) and near D3Mit40 on chromosome 3 (P = 2.2x10^–3) (Fig. 2). No linkage with inflammation or day of paralysis onset that reached the level of suggestive linkage was seen.

View larger version (25K): [in this window] [in a new window]   Fig. 2. Linkage plots for all mice. Dashed lines indicate cut-offs for suggestive linkage (at P = 0.0034) and significant linkage (at P = 0.0001). The y-axis represents the genetic length of each chromosome, with the centromere at the top (filled circle) and relative genetic locations of microsatellite markers indicated. Only chromosomes with loci that show at least suggestive linkage with a trait are shown.

 
Analysis of the inheritance pattern of disease susceptibility revealed that there was a marked difference in the severity of disease when mice that were generated from the mating of female F₁ mice with male B10.S mice were compared with mice that were generated from the mating of male F₁ mice with female B10.S mice. This difference was clearest when weight loss was compared between the two groups (Table 1). Those mice born of a B10.S mother developed substantially greater weight loss after immunization than those born of an F₁ mother (P = 0.0029). This difference was almost entirely among females, where those born of a B10.S mother on average decreased in weight by 3.16%, while those born of an F₁ mother on average increased in weight by 6.78% (P = 1.2x10^–4).

View this table: [in this window] [in a new window]   Table 1. Comparisons of EAE phenotypes in subsets of backcross mice divided according to parents and sex.

 
To control for the parental differences in the mice, linkage analysis was performed on two subsets of mice: mice born of B10.S mothers and mice born of F₁ mothers. Among the mice born of a B10.S mother, a locus at the centromeric end of chromosome 8 (D8Mit3) was significantly linked with paralysis (P = 8.2x1 0^–6) (Fig. 3). This locus also showed significant linkage with inflammation (P = 7.0x1 0^–5). Suggestive linkage with paralysis was found near the middle of chromosome 8 at D8Mit84 (P = 1.6x1 0^–3) and near D18Nds1 on chromosome 18 (P = 1.9x1 0^–3). Among the mice born of an F₁ mother, no linkage with EAE traits was found that increased the level of significance beyond that observed in the analysis of the complete set of mice. Furthermore, no suggestive linkage with the day of disease onset was seen in either subset of mice.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Linkage plots for mice born of a B10.S mother and an F1 father. Dashed lines indicate cut-offs for suggestive linkage (at P = 0.0034) and significant linkage (at P = 0.0001).

 

	Discussion

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SJL/J mice are highly susceptible to the induction of EAE by immunization with PLP in CFA (22). Epitope mapping studies have determined that peptide 139–151 of PLP is a major encephalitogenic epitope for this strain (23,24) and that PLP139–151 is bound by the class II MHC molecule of SJL/J mice, H-2A^s, with strong affinity (25). B10.S mice, which also express the H-2A^s MHC class II molecule, are highly resistant to induction of EAE with either whole PLP or PLP139–151. To determine the basis of this difference in susceptibility to EAE, we crossed the two strains together and backcrossed the F₁ progeny to B10.S to generate mice suitable for genetic analysis. We then tested the progeny for their susceptibility to PLP139–151-induced EAE. Use of synthetic PLP139–151 as the immunogen eliminated differences that might arise from using whole spinal cord homogenate or purified PLP which may contain pathogens as well as additional immunogenic epitopes.

Our previous study on this cross showed that different measures of disease severity, i.e. paralysis, spinal cord inflammation and weight loss, correlated well with each other, but that among animals with the same degree of paralysis, there was a large variation observed in the number of inflammatory foci and the amount of weight loss (3). Considering that EAE susceptibility is a polygenically determined trait, the variation in these sub-phenotypes may reflect a heterogeneous inheritance of subsets of the multiple components that contribute to disease development. Separately analyzing each of the different measures of disease severity, therefore, has the potential to increase the power to dissect out each of the contributing components by focusing on a smaller number of genes influencing the trait. In addition, once a genetic locus that contributes to the disease susceptibility is determined, associating the locus with a specific sub-phenotype of disease can aid in narrowing down candidate genes within the locus. With this in mind, we genetically analyzed the mice separately for each of the different sub-phenotypes of EAE.

Quantitative trait locus analysis also can increase the power of a linkage analysis by correlating the frequency of inheritance of a locus with the magnitude of a quantitative trait. Among the traits that we measured, the weight loss following disease induction was the best suited for analysis as a quantitative trait. As a result of these new analyses, we were able to identify a locus at the telomeric end of chromosome 2 that significantly linked with weight loss. This greatly improved the probability of linkage of this locus with EAE over what had previously been found at this locus when the analysis was done with disease susceptibility as the associated trait. In addition, primarily due to an increase in the number of mice used in the analysis, a locus at the middle of chromosome 8 showed suggestive linkage with paralysis where previously only weak linkage was apparent. On chromosome 3, the locus did not reach significance, but in separate studies we have previously confirmed the presence of a locus affecting EAE severity near the IL2 gene (near D3Mit21) in NOD.B6-Idd3 congenic mice (26) and have other supportive evidence using NOD.B6-Idd17,Idd10,Idd18 congenic mice for a locus near the center of chromosome 3 (unpublished data).

Although this analysis was done with a fairly large number of mice, the linkage scores attained were low, with only one locus reaching the level of significant linkage. This result is similar to what others who have tried to analyze the genetic basis of EAE have found (4,5). While it is possible that this may be due to the number of genetic loci contributing to EAE susceptibility being very large, with each locus only making a small contribution to the overall susceptibility, we also contemplated the possibility that there may be a nonMendelian component of the disease confounding the analysis.

One consideration was that a non-Mendelian factor may be transmitted through the maternal line, such as by genomic imprinting or by an infection passed through the mother's milk. When this was investigated, significant differences in inflammation and weight loss were seen between mice with F₁ mothers and those with B10.S mothers (Table 1). Because the offspring of B10.S mothers had the more severe phenotype, it seems unlikely that an infective factor was passed from the mothers since the opposite phenotype is seen in mother and offspring. Instead, the inheritance pattern fits a model of genomic imprinting where a maternal allele of a gene determining disease severity is silenced. In the offspring of B10.S mothers, the B10.S-derived allele from the mother is silenced, while the SJL/J- or B10.S-derived allele from the F₁ father is expressed, leading to increased disease severity (from the SJL/J-derived allele) in half of the offspring. In the offspring of F₁ mothers, the SJL/J- or B10.S-derived allele from the mother is silenced, while the B10.S-derived allele from the father is expressed, causing no increase in disease severity in the offspring. It is unclear why this difference in the disease severity between the mice from different parents is primarily seen in the females, but it is possible that the disease enhancing factor may be affected by sex hormones or by an X-linked gene. This is particularly interesting in light of the observation that many autoimmune diseases show a sex-associated skewing in susceptibility and merits further study.

On the assumption that a non-Mendelian factor inherited from a particular parent may be altering the disease phenotype, we analyzed the mice separately in two groups according to parentage to eliminate this possibly confounding influence. As a result, in the analysis of offspring of B10.S mothers, a locus near the centromere of chromosome 8 showed significant linkage with both paralysis and inflammation, and a locus on chromosome 18 showed suggestive linkage with paralysis.

We have now identified two loci that are significantly linked with EAE traits and have uncovered a previously unknown inheritance pattern of EAE susceptibility. The linkage of several other loci that may play a role in EAE susceptibility has also been strengthened. An interesting result of analyzing the genetics of EAE based on sub-phenotypes is that disease-associated weight loss appears to be controlled by loci different from those associated with paralysis, suggesting that weight loss may not be simply a direct result of paralysis (e.g. due to an inability to reach for food and water), but may more likely be a result of a loss of appetite or wasting associated with the inflammatory response. This is consistent with our observation and previous reports (17) that the initiation of weight loss generally precedes the onset of paralysis by 1–3 days. It is known that the administration of inflammatory cytokines such as IL-1, IL-6 or tumor necrosis factor induces anorexia, weight loss and protein wasting (19–21). It is possible that following immunization, susceptible mice generate an inflammatory response that results in either a local or systemic release of inflammatory cytokines, which in turn results in the observed weight loss. At the telomeric end of chromosome 2, where the strongest association was found with weight loss, are two genes are present that encode molecules that are known to play a large role in the generation of an immune response and the release of cytokines, the genes coding for CD40 (27) and NFAT-1 (28). We are currently investigating whether polymorphisms exist in these candidate genes that can account for the differences seen between the two strains. With respect to the locus at the centromeric end of chromosome 8 that appears to be involved in paralysis and inflammation, possible candidate genes include scat (a mutation causing severe combined anemia and thrombocytopenia, the phenotype of which only occurs in offspring of mothers with at least one wild-type allele) (29), mnd (a mutation causing motor neuron degeneration) (30) and Plcd (phospholipase C, an enzyme involved in intracellular signaling pathways) (31).

The identification of loci, and ultimately the genes, controlling EAE will help to clarify the pathogenic mechanisms of this disease. Since identical or analogous mechanisms may be involved in human immune responses, this information may lead to novel approaches to the treatment of multiple sclerosis and other autoimmune diseases.

	Acknowledgments

 
This work was supported by grants from NIH (RO1 AI44880, RO1 NS 30843, RO1 NS 35685, PO1 AI 39671 and PO1 NS38037) and National Multiple Sclerosis Society (RG2571 and RG 2320).

	Abbreviations

 

EAE experimental autoimmune encephalomyelitis

CNS central nervous system

LRS likelihood ratio statistic

PLP proteolipid protein

	Notes

 
Transmitting editor: A. Cooke

Received 16 May 2000, accepted 26 October 2000.

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