The Epidemiology of Multiple Sclerosis: Clues to the Etiology of a Mysterious Disease

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1 The Epidemiology of Multiple Sclerosis: Clues to the Etiology of a Mysterious Disease STUART D. COOK Department of Neurosciences UMDNJ-New Jersey Medical School Newark, New Jersey The etiology of multiple sclerosis (MS) has not yet been defined. Based on analysis of prevalence, incidence, and migration studies, it is likely that one or more environmental factors, probably infectious in origin, initiates the disease. Canine distemper virus appears to be an attractive candidate in this regard. Because the risk of acquiring MS is greater in multiplex families (particularly in identical twins), certain ethnic and religious groups, and individuals with the human leukocyte antigen DW2, genetic predisposition is an important determinant in disease expression. NEUROSCIENTIST 2: , 1996 KEY WORDS Multiple sclerosis, Epidemiology, Etiology, Canine distemper Multiple sclerosis (MS) is an acquired inflammatory disorder of the central nervous system (CNS) characterized pathologically by disseminated foci of demyelination with relative preservation of axons. The disease can occur in childhood or late adulthood but usually begins between 10 and 59 years of age. The predilection for MS lesions to involve motor, sensory, autonomic, and integrative pathways typically results in some combination of spastic weakness, impaired sensation, visual loss, tremors, ataxia, incontinence, and occasionally altered cognitive functions. No pathognomonic laboratory test exists for diagnosing MS; however, magnetic resonance imaging (MRI) scans of the brain and spinal cord are extremely useful for detecting the characteristic white matter abnormalities, many of which are not clinically expressed. Sensory system involvement can often be documented with abnormalities in visual, auditory, and somatosensory evoked potentials. In the majority of patients, cerebrospinal fluid (CSF) examination reveals a selective increase in IgG levels, free immunoglobulin light chains, and bands of restricted electrophoretic mobility in the &dquo;(-globulin region indicative of an intense immune response to an as yet undefined host or foreign antigen. The prognosis is extremely variable and often unpredictable. The early course of MS is usually associated with exacerbations and remissions. Subsequently, remissions may be subtotal, and progressive neurological deterioration often ensues. No curative therapy is presently available, although in recent years, interferon beta (IFN-~3) and other immunomodulating regimens have been shown to have a palliative effect on the disease course (1). Address reprint requests to: Stuart D. Cook, M.D., Department of Neurosciences, UMDNJ-New Jersey Medical School, 185 South Orange Avenue, Newark, New Jersey The etiology and pathogenesis of MS are unknown; however, cumulative evidence suggests that one or more environmental factors, probably infectious in nature, triggers immune mediated demyelination in genetically susceptible individuals (2). It is the purpose of this review to analyze this evidence and to propose a plausible hypothesis as to the cause of this enigmatic disorder. Geographic Heterogeneity in the Prevalence of MS MS occurs more commonly in women and in individuals of white European ancestry than in men, Native Americans, African Americans, Asians, and certain ethnic groups (3-5). Prevalence studies indicate a crude northsouth and perhaps a lesser west-east gradient for MS in North America, with MS more common in Canada and the northern United States where prevalence rates as high as 150 to 200 per 100,000 population have been recorded compared with those of the American south, which are generally less than 30 per 100,000 (Fig. 1) (3-5). Kurtzke (3, 4) concluded from a large U.S. veterans cohort study that young white women had twice the relative risk for developing MS as white men, who had twice the risk of African Americans. Each of these groups showed a north-south risk gradient (3, 4). MS risk was also increased for those with more education, urban background, and higher socioeconomic status. In Europe, the occurrence of MS less predictably relates to latitude, although higher prevalence rates are observed in Great Britain and Scandinavia (75 to 300 per 100,000) than in countries bordering the Mediterranean (5 to 70 per 100,000) (6). In some parts of the Southern hemisphere, the north-south gradient is reversed, with a sevenfold higher prevalence of MS in Hobart (southern Australia) than in Queensland (northern Australia) (7) and a threefold higher rate of MS in Otago (southern 172

2 Fig. 1. Worldwide distribution of MS as of Areas of high frequency (prevalence rates of 30 or more per 100,000 population) are indicated by blue areas, those of medium frequency (prevalence rates of 5 to 29 per 100,000) are indicated by stippled areas, and those of low frequency (prevalence rates of less than 5 per 100,000) are indicated by hatched areas. White areas indicate regions for which there were no data. South American frequencies are tentative values. Reprinted with permission from (2). New Zealand) than in Waikato (northern New Zealand) (8). A relatively low prevalence of MS has been reported in most of Asia, despite latitudes similar to areas of higher MS prevalence in Europe and North America, as well as in Africa and South America. Unfortunately, inherent difficulties with the accuracy of case ascertainment make most of the studies from Africa and South America less than definitive. Nevertheless, the lower rate of MS observed in African Americans and Asians, compared with that of whites in the United States, is consistent with the alleged low rates of MS in these racial groups in their countries of origin. Many other exceptions to the relationship between MS prevalence and latitude have been described, and remarkable differences in MS experience have occurred in populations in close proximity, most notably the extraordinarily high rate of MS in Orkney and Shetland (9) relative to the Faroe Islands (10), where MS was allegedly nonexistent before World War II, and the almost 10-fold higher prevalence rate in Sicily relative to Malta (5). Although one can question the validity of prevalence rates in some studies because of suboptimal case ascertainment (attributable to selective migration or immigration, mistaken diagnosis, or omission of mild cases) or challenge comparative prevalence data on the basis of differences in epidemiologic methodology, there seems little doubt about the global pattern of MS, if not the actual numbers. A similar but not identical north-south gradient has been described for insulin-dependent diabetes mellitus, a genetically determined autoimmune disorder of pancreatic beta cells (11). As with MS, higher rates of insulin-dependent diabetes mellitus are found in whites of northern European descent than in African Americans, Native Americans, and Asians, and environmental factors are thought to play a prominent role in disease expression. Others have pointed out similarities in the pattern of MS prevalence to that of poliomyelitis (12), infectious mononucleosis (13), hepatitis B (13), prostatic and colon cancer, dental caries, and Parkinson s disease (14), disorders in which varied genetic and environmental influences have been implicated. In considering possible explanations for the worldwide distribution of MS, Lauer (15) has meticulously analyzed the published prevalence studies assessing the possible role of geoclimatic and sociocultural environmental factors. Among the variables analyzed were geographic relief characteristics; natural radiation; climate; geologic features, including water and soil; forestry; industry; diet; occupation; housing; religion; and agriculture. On the basis of univariate and multivariate analysis, Lauer found a highly significant relationship be- 173

3 Table 1. MS Case/Control Ratios (Actual Numbers) for All White Veterans of World War II or the Korean War by Residence at Birth or at Entry into Active Dutya amodified with permission from (4) tween MS rates and low temperature and humid winter conditions. Because similar climatic conditions are reported for nonspecific respiratory infections, Lauer suggests that climatic factors might be operating through this mechanism. Although Lauer found other associations besides weather, including meat and dairy food consumption, these factors do not appear to be biologically of primary importance. In contrast, Bulman and Ebers (16) note the crude correlation between disease distribution and density of populations derived from northern white European, particularly Scandinavian, ancestry, but this correlation seems imperfect for Australia and New Zealand. Consistent with hereditary determinants, Hillert (17) has pointed out the worldwide association between MS and the human leukocyte antigen DW2 (DR15, DQ6), with the sole exception of Sardinia. Collectively, these findings imply that hereditary factors influence the risk of acquiring MS. In summary, the most likely conclusion from prevalence studies is that both genes and environment are implicated in MS causation. Migration Studies: A Natural Experiment Migration can be considered a natural experiment for determining the relative effects of genetics and environment in a disease of unknown etiology (18). If MS is a disease of place, risk might change with migration; if MS is a disease of person, specific environmental exposure makes little difference in susceptibility and risk should remain relatively constant. However, migrant studies are also subject to many biases, including the sometimes small number of immigrants and the difficulty in accurately determining the number of affected individuals and the size of the population at risk, as well as potential racial, ethnic, and sociocultural differences between migrants and nonmigrants (5, 19, 20). Despite these concerns, studies on migration generally support an effect of environment on MS susceptibility. Perhaps the most robust evidence for such an effect comes from Kurtzke and his collaborators (3, 4). In a retrospective study of 5305 U.S. veterans of World War II and the Korean War who subsequently developed MS and controls matched for age and date of entry into military service, Kurtzke et al. noted a strong north-south gradient for MS. Additionally, a highly significant decrease in risk for developing MS was found for whites moving southward between birth and entry into service, as well as an increase in risk for moving northward from the middle-tier states (Table 1) (3, 4). Kurtzke has also shown that north-south risk rates for MS equalize when death rates are determined for those who were bom in the south but died in the north and those who were bom in the north but died in the south. Similarly, a highly significant decrease in MS mortality in immigrants who were bom in Great Britain but died in Australia compared with MS mortality in Great Britain has been shown (21 ). In this study, mortality rates for native Australians and immigrants from Great Britain were quite similar. None of these relationships would be expected if risk for MS were determined solely at or before the time of birth. Studies of migrants from high-prevalence areas to South Africa (22) and Israel (23) suggest that the risk for acquiring MS remains unchanged for those who move after age 15 but decreases for those who move at an earlier age. This result supports the popular concept that MS is commonly acquired in adolescence but is not or their expressed until later in life. Conversely, migrants offspring who relocate from low- to high-risk areas can, but do not always, increase their risk of acquiring MS. For example, migrants to Great Britain from the West Indies, Africa, and Asia maintain their low risk for MS, but their children assume the higher risk of the local white English population (24). A recent Israeli study by Kahana et al. (25) is also informative in this regard, demonstrating that the MS incidence rate among secondgeneration African-Asian Jews may be higher than that of their parents but remains lower than that of secondgeneration Ashkenazi Jews. In contrast to the results of these studies, other migrant racial groups have been reported to maintain low MS incidence rates, despite living in areas of high MS prevalence (5, 26). Although migration studies have been criticized on the basis of potentially confounding factors discussed previously, little direct evidence shows that they are invalid (19, 20). Therefore, it seems almost certain that environmental factors can influence MS risk. Incidence Studies Suggest Environmental Factors If MS were solely a genetic disorder or if multiple nonspecific environmental agents initiated the disease process, MS would be a stable endemic disease in the population, with incidence rates remaining relatively 174

4 Box 1: Definition of Epidemiological Terms Incidence: The number of new cases of a disease in a given population in a defined period time. Prevalence: The number of existing cases of a particular disease in a defined population at a given time. Migrant: One who moves from one place to another. Immigrant: One who permanently migrates from one country to another. Mortality rate: Number of individuals expiring with a particular disease in a given population in a defined period of time. Risk: The odds of a defined cohort developing an illness. Relative risk: Ratio of the risk of one cohort developing an illness compared with that of another. Cluster: An excessive number of patients with an illness in a defined population relative to the expected number of patients. Not all clusters are biologically meaningful. constant. Conversely, if one or only a few environmental factors were crucial and if exposure to these factors were altered, MS incidence would be expected to reflect a parallel change, with a delay equal to the incubation period of the disease. However, in assessing incidence data, optimal case ascertainment and deployment of similar rigorous methods in comparative studies are essential. In addition to the inherent difficulty with accurate case ascertainment, specific problems exist for incidence studies (5,19) (Box 1). The often long delay before the diagnosis of MS can be established and the outward migration or death of symptomatic but undiagnosed individuals can lead to a falsely low incidence rate. Inclusion of immigrants or natives whose disease began elsewhere could lead to the opposite conclusion. Further, it is often difficult to be sure of the precise year of MS onset. These issues are particularly important to consider when analyzing incidence in geographic areas with small or highly mobile populations. Because of these difficulties, worldwide patterns of MS have generally been described on the basis of prevalence rather than incidence, whereas incidence studies have usually been conducted to assess whether change in disease frequency has occurred in a defined area. As might be expected, many reports of upward and, less commonly, downward fluctuations in MS rates have been published, although the significance of these changes is disputed. For example, MS incidence rates have increased in many areas undergoing repeated surveys, including Rochester, Minnesota, Finland, Italy, and Western Norway (2). Although these changes have generally been attributed to better case ascertainment in more recent studies, the possibility that a true increase in incidence has occurred has been suggested (2, 28). For example, in Western Norway an increase in MS incidence (29) was correlated with an increase in disability pensions and in the MS mortality rate, a constellation of measurements claimed unlikely to be explained solely by better case ascertainment (30). Conversely, a significant downward trend in MS incidence was observed in Denmark from 1952 through 1967 (31 ). Although the decline was calculated from the national Danish Multiple Sclerosis Registry, the registry was not established until 1956, implying that early data on incidence and prevalence may have been incomplete. A difference in case ascertainment methodology might also explain the decrease in MS found in Gothenburg, Sweden, from 1974 to 1988 as compared with the 1950 to 1964 (32) rate. Alternatively, these observations may be real and consistent with an environmental alteration not yet identified. Disease clustering is a special situation where the number of patients with a particular disorder exceeds expectations (20). However, problems exist as to what change constitutes a true cluster and not one occurring because of chance alone (33). At a minimum, true clustering of MS would provide proof of environmental influences but also, more importantly, valuable insight as to the cause of the disease (33). With regard to MS, controversial clustering has been described in some areas, including the Faroe Islands (10), Iceland (34, 35), Newfoundland (36), Key West (37), and Western Norway (29). Unfortunately, no common theme can explain all clusters, although in some cases, antecedent canine distemper epidemics in the dog population allegedly had occurred (38). Genetic Factors Also Play a Role Multiple lines of evidence indicate that the seed, in addition to the soil, is important in predisposition to MS. Assessment of MS concordance in twins is quite illustrative in this regard. In most studies, the concordance rate for MS is six to eight times higher in identical than in nonidentical twins, and the concordance rate in nonidentical twins more closely resembles the rate found in non-twin siblings (5). For example, in the Canadian twin study, 30.8% of monozygotic and 4.7% of dizygotic twins developed MS, compared with 5.1% of non-twin siblings (39). An identical rate of MS in non-twin siblings and dizygotic twins would be expected on the basis of genetic determinants but not environmental exposure because twins are more likely than siblings of different ages to share a common exposure. For second-degree relatives, the risk for MS falls to 1% but is still higher than the 0.1 to 0.2% found in the general population (40). Considering all relatives, a positive family history of MS was found in almost 20% of patients in the Vancouver study (40). However, lower rates of familial MS have been found in other areas, particularly where MS is less common. Analysis of the age of onset and year 175

5 of onset in MS sibling pairs is also revealing, because the former correlated more closely than the latter (41, 42). This correlation would be predicted for a genetically determined disease, whereas the opposite would be expected for an environmental disorder. The most plausible explanation for these varied observations is that susceptibility to MS is genetically determined. Because MS is common in some racial and ethnic groups and rare in others, attempts have been made to explain these differences on the basis of environmental, cultural, and genetic factors. Initially, genetic studies focused on candidate genes, particularly those involved in the immune response. Population and familial studies demonstrated an association but not linkage between MS susceptibility and the MHC Class 2 phenotypes DR15 and DQ6 (43). To date, this haplotype is the only one clearly related to MS; however, HLA is thought to contribute only about 10% to overall susceptibility (44). As with other autoimmune diseases, only a minority of patients with the appropriate HLA haplotype develop MS, and MS can occur in the absence of DR15 and DQ6. Attempts to link MS to other candidate genes, including alpha and beta T cell receptors, immunoglobulin heavy chains, myelin proteins, and complement and tumor necrosis factor have been negative, controversial, or inconclusive ; therefore, several laboratories have recently changed strategy and are now conducting random searches for gene linkages in multiplex MS families, using representative spaced markers for the entire genome (43). Based on genetic contributions to other autoimmune diseases, the observed MS inheritance patterns, and ongoing genetic studies, it has been hypothesized that multiple gene loci will be shown to be responsible for MS susceptibility but probably not for MS cause (5, 40). The Viral Hypothesis Although there is little doubt that genetic susceptibility determines who is capable of acquiring MS, there is equally strong, albeit also indirect, evidence that one or more environmental factors are necessary for the disease to be expressed. The evidence for an environmental trigger has been summarized as follows (45): the worldwide pattern of MS is both nonrandom and disease specific, but altering residence can alter disease prevalence in migrants or their offspring; the concordance rate for MS in monozygotic twins is relatively low (30.8%), even with brain MRI studies and long-term follow-up (39). A similar concordance rate in monozygotic twins has been reported for paralytic poliomyelitis, a disease of known viral etiology (46). Spontaneous CNS demyelination occurs in animals and humans after viral infections, which clinically may be monophasic, recurrent, or progressive (45). High antibody titers to several infectious agents, and particularly to measles virus, are present in the CSF and serum of patients with MS (47). Patients with MS have childhood infections, including measles, at a later age than controls in regions of high MS prevalence (48). Controversial time clustering of MS has been reported in several distinct populations (10, 29, 34-37). Although some hypotheses have been presented to explain these varied observations, the most universally accepted and most plausible is that an undefined infectious agent(s) triggers MS. The major disagreement is whether many nonspecific pathogens are capable of initiating the disease or whether classic MS is caused by one or just a few agents perhaps sharing common immunopathogenic mechanisms (19). The occurrence of spontaneous autoimmune diseases, including experimental allergic encephalomyelitis (49) and diabetes mellitus (5) in transgenic inbred or genetically homogeneous animals, can be influenced by changing environmental conditions in which the animals are housed, suggesting to some the likelihood of multiple random inciting factors. Others believe the unique worldwide pattern of MS, which differs from the pattern of most autoimmune diseases, the effect of migration in altering risk, the characteristic adolescent or young adult age of maximal susceptibility, twin studies, and the apparent absence of MS in the Faroe Islands before 1943 are quite consistent with a single or relatively few specific causative agents (2, 45). An example of a single-agent, geographically nonrandom, human organ-specific autoimmune disease is streptococcal-induced rheumatic heart disease, thought to be triggered because of molecular mimicry between bacterial M protein and myosin (50). Although many infectious agents have been proposed to cause MS, no agent has been consistently isolated from MS tissue, and neither viral genome nor viral protein has been definitively identified in MS brain specimens, even with exquisitely sensitive techniques, such as polymerase chain reaction (PCR) (45). Nevertheless, some viral and nonviral organisms remain plausible as candidates because they can cause demyelinating disease in animals or humans or because antibody titers to these agents are higher in MS patients than in controls (45). Other pathogens are consistent with the unusual worldwide pattern of MS. For example, in underdeveloped countries where sanitation is suboptimal and MS is uncommon, exposure to some viruses may occur early in life, whereas in North America or Europe, exposure to these same agents may occur at a later, perhaps more vulnerable age (12, 13). The worldwide patterns of paralytic poliomyelitis, infectious mononucleosis, and hepatitis B are thought to best fit with such a mechanism (12, 13). However, if this were true for MS, risk should increase with migration from high- to low-prevalence areas because greater exposure to the putative agent would be expected to occur, which is the opposite of what is observed. In contrast, some agents may be more prevalent in cold, humid climates, or even if the virus is equally prevalent in warm and cold climates, cultural 176

6 differences might lead to greater exposure to the agent. For example, greater exposure to indoor dogs has been hypothesized to explain the higher rate of human hydatidosis in colder parts of Kenya where dogs are kept indoors than in warmer regions where dogs are more commonly outdoors (51). If MS is initiated by a virus, several major mechanisms could induce demyelination (45). The virus could actually exist in the brain or other host tissues but avoid detection. Failure to identify an infectious agent to date in MS tissue should not be interpreted as proof that a virus is not present (45). Several RNA viruses, including canine distemper virus (CDV), a morbillivirus of dogs; JHM, a mouse coronavirus; and Theiler s murine encephalomyelitis virus can induce demyelinating diseases in which viral genome may remain detectable in brain. Subsequently, a brain viral infection could cause de- an immune mechanism. myelination directly or through In this situation, it should be possible to identify viral genome or expressed proteins with in situ hybridization, PCR, and immunohistochemical techniques. Alternatively, infectious agents may trigger an autoimmune process but not be present in the host target organ when disease is clinically apparent (45). This can occur through release of sequestered antigens because of direct viral injury or through an immunologic reaction against shared structural proteins between the infectious agent and the brain (molecular mimicry) (52). If such is the case in MS, linking a virus to the disorder may be very difficult and will probably require a combination of serological and epidemiologic evidence of infection (45). The CDV Hypothesis It is beyond the scope of this article to consider all of the infectious agents postulated to cause MS. Several excellent reviews have covered this topic recently (45, 53). Instead, this article focuses on CDV, the measleslike morbillivirus of dogs that has become increasingly attractive as a candidate for triggering MS (45). Morbilliviruses cause spontaneous CNS inflammatory or demyelinating disease in a variety of species, including human, seal, porpoise, dog, tiger, ferret, lion, and raccoon (45). CDV is one of the most neurotropic forms of morbillivirus, with some strains causing demyelination in up to 90% of infected dogs (54) compared with a 0.1 % rate of measles after encephalomyelitis in humans (53). CDV can jump species, causing CNS disease in a wide range of animals, including primates (55), and CNS demyelination with an acute or relapsing-progressive course in dogs (56). Animals may have varied neurological abnormalities, including seizures, myoclonus, ataxia, paralysis, tremors, or optic neuritis. As in MS, dogs with CDV infections of the CNS often have an increase in CSF IgG levels (57). In dogs with a relapsing-progressive course, lesions similar to MS plaques can form in the CNS (45) and are usually multifocal and periventri- Table 2. Dog Ownership in MS Patients and Age- and Sexmatched Controls before First Symptoms of MSa amodified with permission from (27). cular in location (45). Although the virus is usually found in the acute demyelinating brain, it may be difficult to detect or be undetectable by conventional techniques in the relapsing-progressive form of this disease (45). It is generally believed that the acute demyelinating lesions result from the direct lytic effect of CDV, whereas the chronic demyelinating lesions are probably immune mediated (45). MS, Dogs, and CDV Exposure Although limited data are available on dog ownership, dog density (and MS prevalence) is greater in Europe and North America than in India (Bansil et al., in preparation) and probably China and Japan (45). Studies in the United States have shown a latitudinal relationship of dog demographics, with a significantly higher proportion of dogs kept indoors in the colder northern than in the warmer southern United States and intermediate rates found in the midtier states (58). This latitudinal distribution of indoor dogs is consistent with the distribution of MS in the United States. If MS is a zoonosis caused by CDV, greater patient exposure to dogs before onset of neurological symptoms might be expected. However, the high level of human exposure to dogs in the Western world (60-80%) makes retrospective, small case control studies relatively insensitive for testing the CDV-MS hypothesis (45). Nevertheless, at least nine retrospective case control studies have shown significantly more dog exposure among pre- MS patients (Table 2) (45). Although fixrther studies failed to show this relationship, none showed significantly more dog exposure in controls. Although dog exposure per se would be expected to be important if the CDV-MS hypothesis were correct, the more important relationship would be exposure to dogs with a CDV infection (45). In this regard, three studies showed significantly greater exposure to dogs 177

7 with a distemper-like illness before the onset of MS, and three others noted more exposure without achieving statistical significance. Unfortunately, retrospective studies such as these can be criticized on the basis of the long time elapsed before onset of illness in many patients and the possibility that MS patients may be biased toward recalling neurologically ill pets (45). Like measles, CDV occurs in all climates but peaks in colder, damper months--conditions conducive to greater human contact with indoor ill dogs (45). Also like measles, CDV can occur in explosive epidemics. Epidemics of CDV have been followed by significant but controversial increases in MS incidence, and controversial time clusters of MS have been preceded by outbreaks of CDV in the dog populations of Newfoundland (36), Key West (59), Sitka (60), the Faroes (61), the Orkneys (62), and Iceland (35), although Benedikz (63) disputes the existence of MS epidemics in Iceland, Poser (64) denies an epidemic of MS in the Faroes, and Kurtzke (65) denies an epidemic of CDV in the Faroes. Kurtzke s conclusion can be challenged because his studies were based on retrospective analysis of interviews and questionnaires 30 to 40 years after the CDV outbreak and because they were refuted by the recollections of the Faroe veterinarians, if not by their written records. According to Kurtzke, there is an imperfect correlation between the location of troops and the residence of MS patients; however, one cannot be sure of movements of dogs or the native Faroese, or the degree of contact between them. Further, the serological studies for CDV antibodies in the Faroes, conducted by our group and used by Kurtzke to refute the CDV-MS hypothesis, were unable to distinguish between CDV and measles antibodies. Our conclusion, contrary to Kurtzke s, is that a CDV outbreak did occur in the Faroes, with the first case of CDV occurring 1 to 2 years before the first case of MS. CDV Serology Recently, our group has carried out enzyme-linked immunosorbent assays (ELISAs) for canine distemper-specific IgG antibodies in MS and control sera (45, 66). In this assay, three linear peptides of CDV, each 15 to 16 amino acids in length, corresponding to the three most hydrophilic regions of the CDV H protein, were synthesized and used as antigens (45, 66). Two of the three CDV-H peptides had one amino acid sequence in common with the corresponding measles virus peptides, whereas the third had none (45, 66). Because the assay did not detect CDV antibodies in measles-hyperimmune animals or in patients with acute or chronic measles infections-all with high measles antibody titers-but did detect CDV antibodies in animals vaccinated or infected with distemper, we were able to clearly distinguish between measles and CDV antibodies for the first time. We then tested human sera for CDV-specific antibodies. Sera from 55 MS patients showed significantly higher IgG antibody titers to all three CDV peptides compared with those of 125 age-sex-matched sera from a variety of controls, including normal individuals; patients with other neurological diseases; and patients with infectious, autoimmune, or inflammatory disorders (66). Most of the CDV-positive sera had elevated titers to all 3 CDV peptides. Although only 29% of MS patients had antibody levels >2 SD above controls, there was 70% specificity of high-titered CDV sera for MS (66). MS sera did not differ from control sera in ELISAs against either a corresponding synthetic measles virus peptide or a polio peptide (Rohowsky-Kochan et al., in preparation), and CDV-positive and -negative sera did not differ in IgG levels, again implying specificity of the assay for CDV (45, 66). It is unlikely that our assay detected antibodies to other pathogens because a search through peptide data banks revealed no common protein-sharing amino acid sequences with all three of our CDV H peptides (45, 66). The actual rate of seropositivity to CDV may be higher than detected because antibody titers may have fallen if contact with CDV occurred many years earlier (45, 66). Further, a linear peptide may not be as antigenic in vitro compared with the same peptide as part of a complex protein-lipid-carbohydrate antigen in vivo (45, 66). Lastly, CDV titers may be lower in patients who had a measles infection before a CDV infection, just as humans usually have the highest influenza antibody titer to the strain of influenza virus causing their initial infection (theory of original antigenic sin) (45, 66). On the basis of our serologic studies, humans appear to be infected by the neurotropic CDV. Moreover, we believe CDV is probably the leading candidate agent in triggering MS currently because CDV titers are highest in MS patients; CDV can cause a clinical and pathological disease similar to MS in animals; multiple studies have found dog and CDV exposure to be higher in MS patients; and CDV can plausibly explain some of the demographic features of MS and, perhaps, some of the controversial time clusters. Criticism of the CDV-MS hypothesis includes the failure to identify the CDV genome or antigen in MS brain tissue (67); the failure of MS to decline, despite the increased CDV vaccination of dogs (29); and the persistence of MS in populations where dogs are thought not to have been exposed to CDV (27, 63). However, defenders of the CDV-MS hypothesis might point out that vaccine strains of CDV do not readily detect wild virus by in situ hybridization (although they may by PCR) (68); sporadic cases of CDV are still common in many areas; major epidemics of CDV have occurred in Switzerland and Australia in the last decade; and vaccination or revaccination of dogs in the United States is relatively low (estimated 38%), despite veterinarian recommendations for annual revaccination. In the final analysis, whether CDV actually causes MS or is a major cause of MS remains to be determined by further ser- 178

8 oepidemiologic studies, additional attempts to identify the CDV genome in MS tissues, and demonstration of decreases in MS incidence following widespread dog vaccination with this virus. Conclusions Evidence has been presented supporting the role of susceptibility genes in combination with unknown environmental factors in the causation of MS. It seems probable that multiple genes account for susceptibility, which makes it unlikely that gene therapy will be available in the near future to prevent or alter the course of the disease. If MS is triggered by multiple random environmental factors, it is also very unlikely that changes in environment will have a major impact in decreasing MS incidence. On the other hand, if MS is caused primarily by one agent and tne evidence is not inconsistent with this possibility, then the exciting prospect exists that disease expression can be modified by the development and widespread deployment of an effective vaccine. References 1. 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