J R Coll Physicians Edinb 2002; 32:244–265
P.O. Behan, Professor Emeritus of Clinical Neurology and
A. Chaudhuri, Senior Lecturer in Clinical
Neurosciences, Department of Neurology, Institute of Neurological Sciences, Glasgow University,
Scotland; B.O. Roep, Associate Professor in the Immunology of Diabetes, Department of
Immunohaematology and Blood Bank, Leiden University Medical Center, Leiden, The Netherlands.
The fact that an opinion has been widely held is no evidence whatever that it is not utterly absurd; indeed in view of the silliness of the majority of mankind, a widespread belief is more likely to be foolish than sensible.SUMMARY
Bertrand Russell - Reproduced with kind permission from ‘Marriage and Morals’ The Charlestown Gazette; 1950.
Multiple sclerosis (MS) is a chronic disease of unknown aetiology that affects the human central nervous system (CNS). Its pathology is characterised by areas of myelin loss in a periventricular and perivenular distribution in association with conspicuous astrocytic proliferation and a variable degree of neuronal and axonal damage. Only a proportion of lesions are clinically eloquent. The principal determinant of long-term MS disability is neuronal degeneration and this may be extremely variable. The presence of mild scant lymphocytic infiltrates in the demyelinating lesions has been generally interpreted as the evidence of an inflammatory autoimmune process. Because specific T-cell mediated autoimmunity can be reproduced in animals after myelin protein sensitisation (Experimental Allergic Encephalomyelitis (EAE)) it has been assumed (but never proven) that a similar T-cell driven immune mechanism is responsible for demyelination in MS.
In this review the literature for evidence of autoimmunity in the disorder is analysed critically. In contrast to the accepted theory, the human counterpart of the experimental autoimmune demyelinating disease, EAE is not MS but a different demyelinating disorder, i.e. acute disseminated encephalomyelitis (ADEM) and acute haemorrhagic leukoencephalitis (AHLE). Extrapolation of EAE research to MS has been guided largely by faith and a bland acceptance rather than sound scientific rationale. No specific or sensitive immunological test exists that is diagnostic of MS despite the extensive application of modern technology. Critical analysis of the epidemiological data shows no association with any specific autoimmune diseases. Geographic factors and age at development suggest an early onset possibly dependant on environmental influences.
Certain neurological diseases are, however, found in association with MS, namely hypertrophic peripheral neuropathy, neurofibromatosis-I, cerebral glioma, glioblastoma multiforme and certain familial forms of narcolepsy. These share a common genetic influence mainly from genes on chromosome 17 affecting cell proliferation. Immunosuppression has failed to have any consistent effect on prognosis or disease progression. The available data on MS immunotherapy are conflicting, at times contradictory and are based on findings in animals with EAE. They show predominantly a 30% effect in relapsing/remitting MS that suggests a powerful placebo effect.
Our studies allow us to offer an alternative hypothesis of pathogenesis. We suggest that MS is a neurodegenerative and metabolic disorder with a strong polygenic influence, the predominant genes being on chromosome 17, and in conjunction with environmental factors and endogenous sex hormones. The principal cellular abnormality appears to occur in the astrocyte and this gives rise to disruption of the blood-brain barrier with secondary metabolic changes in the myelin. The process is generalised throughout the CNS, the plaques being focal areas of increased tissue damage. Our data would argue that primary progressive MS is the prototype disease and that what is needed in future research is a new approach based on the firm established facts that now exist.
The cause of MS is unknown, as is the exact pathogenesis of the disorder.1 This point needs to be stressed as there is a common recurring statement in virtually every paper and textbook that MS is of autoimmune aetiology. Multiple sclerosis is clinically a heterogeneous condition and to date still defies both clinical and exact definition.2 Sadly, these factors may be responsible for the minimal progress that has been made towards understanding its aetiology and pathogenesis or in developing a rational mode of therapy. At present, two main theories are proffered as to its causation, namely that it may be due to a viral infection or to a viral infection which instigates autoimmune mechanisms. No firm evidence exists to support either of these contentions. Epidemiological data also suggest that exposure during childhood to some environmental agent may be the initiating event, but need not be infectious.
Certainly, the occurrence of MS is greater in first, second and third degree relatives of MS patients than in matched control populations. Similarly, the concordance rate is greater in monozygotic as opposed to dizygotic twins. Evidence thus points to it being a polygenic disorder.3
Some studies have claimed to have found an MS susceptibility locus in the Class II region of the major histocompatibility complex (MHC);4 another point used in support of the autoimmune hypothesis but it should be realised that many genes also in this region have nothing to do with the immune system. The increased recurrence risk within families clearly indicates a role for genetic factors in the aetiology of MS. A number of genes may influence susceptibility to the development of MS and the subsequent course of the disease. Extensive searches across chromosomal candidate regions and whole genome screens have so far yielded few convincing candidates. In common with most other complex traits, no major susceptibility gene has been identified, although several regions of potential linkage have been associated with MS, including the chromosomal regions 1p, 6p, 10p, 17q and 19q.5
As the cause of MS remains unknown, it is important to explore the possible mechanisms of disease pathogenicity that may provide clues to identifying susceptibility loci. Epidemiological studies have identified other diseases associated with MS, namely malignant glioma, neurofibromatosis and hypertrophic peripheral neuropathy. The mechanisms of these diseases are independent. It is intriguing that the gene for neurofibromatosis, a disease characterised by increased incidence of glioma, has been located on chromosome 17q,6 a region which has been previously associated with MS,7 glioblastoma multiforme,8 and Charcot-Marie-Tooth peripheral neuropathy.9
For the past century, the major lines of research in MS have focused on an immunopathological aetiology. This has enormous implications, not only for the future and direction of research, but also in directing treatment. Despite many attempts at immunosuppression, no convincing evidence has been put forward that such therapy has any significant effect on the clinical course of the disease.10 Whilst there are some claims for the possible modest efficacy of ß-interferon, many caveats surround this conclusion,2,11 and it should be realised that even the mechanism of how ß-interferon may work is not understood, although it may have some healing effect on the blood-brain barrier.12 ß-interferon is certainly not a cure, nor does it induce great symptomatic improvement, whilst immunosuppressive therapy has many serious side-effects and complications, including fatality. The consequence of accepting an autoimmune hypothesis may therefore have a very serious outcome in relation to therapy.
In this paper, therefore, we wish to examine critically factual information surrounding MS and to draw attention to the many fallacies that exist regarding it. Since the aim of research is to use unbiased data to direct future studies, an open-minded approach may help to direct future investigations into more fruitful paths.
RELATIONSHIP BETWEEN MS AND EAE
The idea that certain neurological diseases have an ‘allergic’ basis was advanced in 1927 by Glanzmann, as an explanation for the encephalomyelitis that complicates certain viral infections, such as chickenpox, smallpox and post-vaccination.13 Soon after the introduction of the rabies vaccine by Pasteur in 1876, it was noted that several patients developed paralysis and other neurological dysfunctions after receiving the vaccine. Critical observers also noted that often the offending dog that had bitten them was later found not to be rabid. Remlinger in 1928 analysed the clinical details of 1,164,264 patients treated with the Pasteur vaccine and found 529 cases with such neurological complications.14 Attention was directed later as to the possible cause of such neurological complications and at the potential for disease induction by materials in the vaccine other than the virus.
Animals would not tolerate immunisation with brain material and often developed convulsions, paralysis and weight loss.15 Early workers studied possible allergic reactions in the brains of animals, and these studies, coupled with the finding that whole brain extracts could induce encephalitis, gave an enormous impetus to this research. The brains of patients with such ‘neuroparalytic accidents’ were found histologically to differ from those dying of rabies.16 Schwentker and Rivers showed that the poorly antigenic homologous brain inoculum used to try to induce allergic encephalitis could be rendered more antigenic by autolysis or infection; the antigenicity of brain tissue paralleled its myelin content.17 Encephalomyelitis was capable of being induced in monkeys by repeated injections of aqueous or alcohol/ ether extracts of brain,18 with the disease appearing after months of repeated injections. After the discovery of Freund’s adjuvant, EAE could be produced by a single immunisation in guinea pigs, rabbits and monkeys.19,20
Comparative neuropathological studies of EAE drew attention to histological similarities between this experimental model and post-rabies encephalomyelitis in humans.21,22 It is our contention that the extension of this comparison to include the prototype primary human demyelinating disease, MS, is one of the major pitfalls in MS research. Experimental allergic encephalomyelitis is totally different clinically, immunologically and histologically from MS although it does have similarities with other human demyelinating diseases. Experimental allergic encephalomyelitis is one of the best studied organspecific experimental autoimmune diseases in animals.
A voluminous literature has grown up on the immunological findings, the innovative immunological techniques and methods that have been tested or developed using this model. It has been shown that T-lymphocytes sensitised to a variety of different CNS antigens induce disease. Such antigens include myelin-basic protein and its breakdown products, i.e. encephalitogenic proteins and proteolipid protein (PLP) which are all part of the oligodendrocyte/myelin complex. Work on this important model is scientifically important in its own right. It is only when the data gleaned from studying the brains of mice or rats with EAE is extrapolated to humans with MS that the fallacy of such leaps of faith is demonstrable. Every time that a new observation is made on EAE, such as the traffic of lymphocytes within the brains of rats, researchers incorporate these findings into the dogma that similar findings occur in MS.23 However, EAE appears to be identical to post-rabies and post-infectious ADEM.
Acute disseminated encephalomyelitis and its hyperacute form, acute necrotising haemorrhagic leukoencephalopathy (variously known as acute haemorrhagic leukoencephalitis (AHLE)) are inflammatory diseases of the human CNS which occur following rabies vaccinations, other vaccinations, immunisations and after viral infections,24 acute disseminated encephalomyelitis may also occur as a complication of endotoxic shock, severe burns.24, 25 It may also occur where there is damage to cerebral endothelial cells.25 It may be a mild disorder manifest clinically by a little ataxia or can be a devastating or severe disease with death occurring within hours to days.24 Acute disseminated encephalomyelitis and AHLE are part of the spectrum of the same disorder.26 These diseases are often classified as primary demyelinating diseases but in reality they are vasculopathies of the CNS, in which only a tiny area of myelin loss occurs adjacent to the affected vessels and rarely exceeds 1–2 mm in diameter. Acute disseminated encephalomyelitis is similar to EAE and differs entirely in all aspects from MS.22, 27 Comparison of AHLE and ADEM to EAE in monkeys shows that an exact analogy exists between hyperacute EAE in monkeys and between ‘ordinary’ EAE and ADEM, in all clinical, pathological and immunological aspects. 24,27,28
ADEM and AHLE
Abundant immunological and other evidence shows that the lesion in ADEM and AHLE in humans is at the brain/ endothelial cell interface and that any demyelination which occurs is a secondary bystander reaction. These conditions should not be called primary demyelinating diseases since they are severe inflammatory disorders with little or no resemblance to the prototype primary demyelinating disease of humans, MS.22, 27 Acute haemorrhagic leukoencephalitis and ADEM occur in conditions that are known to be pure endothelialopathies. 25
Acute disseminated encephalomyelitis has been known about for nearly 300 years since its initial description by Clifton in 1724 and has been referred to post-infectious, para-infectious, post-vaccinial, perivenous, microglial and post-rabies encephalomyelitis.24 Remarkably, its exact pathogenesis has not been carefully studied when one considers that this form of encephalitis accounts for onethird of all cases clinically diagnosed as encephalitis in the US.29 Acute disseminated encephalomyelitis is related to AHLE;26 indeed, the histological features of both diseases are often found in the same brain. The immunological findings that occur in ADEM, and AHLE particularly, are also found in EAE in monkeys27, 28, 30 and are absolutely different from MS. Their clinical presentation is also different being a monophasic disease.22, 27 Acute disseminated encephalomyelitis and AHLE are only known to occur after predisposing causes, e.g. infection, immunisations, vaccinations or as a complication of endotoxic shock.24, 25
Patients often present with fever, headache, meningism, vomiting and anorexia, rapidly followed by delirium, confusion, stupor and sometimes coma. The differential diagnosis of ADEM and AHLE is that of an acute, infectious meningoencephalitis rather than MS.24,30 In a classical case, there is no difficulty in differentiation from MS because fever, aphasia, meningism, bilateral optic neuritis, stupor and coma are decidedly rare, if they ever occur, in that disorder.31 Pathologically, the brain in ADEM is oedematous and swollen. Histologically, perivenous inflammation occurs with 1–2 mm in diameter of tissue destruction and fragmentation of myelin. The perivascular cellular cuff may extend for a variable distance into the white matter where there is proliferation of microglial cells and astrocytes show swelling of their cytoplasm. Microglial cells become phagocytic and lipid stains reveal that these cells are laden with fat. When the brain is examined in the recovery phase, there is perivenous fibrous gliosis. Some of these perivenous damaged areas may coalesce to form a larger lesion but these changes are entirely different from MS plaques.32 In the perivenous lesions of MS, the highest density of cells is at the edge while in ADEM it is at the centre of the lesion. In ADEM there may be frank fibrinoid necrosis of vessels with exudation of plasma.32 Occasionally glial nodules, known as Babes’ nodes, which are morphological evidence of a pre-existing viral infection may be found. Immunologically, in some cases sensitisation to myelin antigens30,33 may occur and this sensitisation may be involved in directing immunological attack at the blood vessels of the white matter. The degree of sensitisation is modest and may even be secondary. Acute disseminated encephalomyelitis is identical to EAE22, 28 both clinically and histologically.
Experimental allergic encephalomyelitis has been readily induced in a wide variety of experimental animals including non-human primates.24, 28 Clinically, it runs a monophasic course, usually with a dramatic onset of weakness, hind leg paralysis and incontinence of urine in lower animals but often optic neuritis, cerebellar signs and paralysis in non-human primates.24, 28 The disorder clearly involves T-lymphocytes but the histological appearance varies enormously between animal species, and its clinical and histological expression depends on such factors as the type of animal and the route and method of immunisation. Monkeys injected with a small quantity of purified myelin-basic protein in complete Freund’s adjuvant usually develop a mild monophasic clinical illness in which histologically perivenous mononuclear cell infiltrates are found in the white matter, similar to those found in ADEM in humans.22 Repeated injections using the same procedure but with the addition of pertussis bacilli in the adjuvant, or with whole white matter as antigen, will result in an entirely different clinical and histological outcome. These animals will have multiple abnormal cerebral foci, which are frankly haemorrhagic with severe oedema, fibrinoid necrosis of blood vessels within the lesion and a cellular component varying from mononuclear lymphocyte CD4+ cell infiltrates to that of intense polymorphonuclear neutrophil exudates with extravasation of fibrin and serum.23 In our experience of inducing hyperacute EAE in baboons and monkeys, the condition is identical to AHLE in humans. Experimental allergic encephalomyelitis is an intensely aggressive inflammatory disorder in which myelin loss occurs, predominantly as a bystander reaction, in a small perivascular sleeve of tissue as opposed to MS which is characterised by large demyelinating plaques, conspicuous astrocyte proliferation with or without scant lymphocytic infiltration.34–6
Hyperacute EAE, therefore, i.e. the disease that occurs when EAE is induced by routine methods in cats, dogs and monkeys, bears no clinical or histological resemblance to MS. This form of EAE and AHLE in humans is clinically identical with the same findings in the cerebrospinal fluid and in the brain. In EAE in monkeys and AHLE in humans, such intense cerebral oedema occurs that gross morphological displacement of brain structures often occurs along with an increase in water content of the brain of up to 20%.22 If the brain of normal animals is traumatised, it too may develop a cellular infiltrate that is composed of macrophages and T-lymphocytes.37 Concomitant with this perivenous inflammation is the breakdown of the blood-brain barrier.38 The exact mechanism of why this occurs is unknown but may be related to inflammatory mediators released from macrophages and other invading mononuclear cells. Similarly, animals with traumatic damage to the CNS will also show increased cerebral vascular endothelial permeability.39, 40 The breakdown of the blood-brain barrier is the one feature that EAE and post-traumatic lesions of the CNS have in common with AHLE, ADEM and, indeed, MS.
Dissimilarities between EAE and MS
The lesions in EAE are different to those in MS where the characteristic feature is the well-defined edge, clearly differentiating it from that of ADEM and hence EAE. The inflammatory cerebral lymphocytic infiltrates that occur in most cases of MS are usually weak, scant, lack aggressiveness34–6 and, as pointed out by Lumsden, cannot be compared to those of ADEM.35 Mild inflammatory haematogenous infiltrates and oedema which are not consistent pathological findings in MS have been deemed by Lumsden not to be essential morphological criteria.35 He drew attention to the self-propagative character of the MS lesion which showed variable discontinuous sleeves of demyelination surrounding blood vessels which he regards as the essential distinction between the more regular and uniform perivenular lesions of ADEM and EAE.35
Experimental allergic encephalomyelitis has been used as a model for MS and it is the best studied organ-specific autoimmune disease: it is not acceptable as a model for MS for many reasons, some of which have already been alluded to. Clinically, it is a monophasic disease, the lesions in the brain and spinal cord are vasculitic and the immunological findings are not found in MS. Treatment protocols in EAE are many but those used in humans, based on the findings in animal EAE, have singularly failed in alleviating the symptoms and signs of MS. Our contention is that it is inaccurate to extrapolate the findings in this putative animal model to the pathogenesis of human MS.
Experimental allergic encephalomyelitis can be induced in a large number of susceptible animals by injection of whole brain, pure white matter, myelin-basic protein or PLP in complete Freund’s adjuvant. In susceptible and inbred animal strains, EAE can be induced by cell transfer, and this proves it to be the prototype T-cell mediated autoimmune disorder created artificially for experimental purposes. The encephalitogenic factor responsible for disease induction has also been characterised and manufactured synthetically in a number of species.
Experimental allergic encephalomyelitis is under genetic influence, related to the major histocompatibility complex- (MHC) Class II antigens and to other gene loci.41, 42 Whilst examination of the model in strain 13 guinea pigs and in DA rats (a genetic species of rat which is similar to strain 13 guinea pigs; in both strains it is easier to induce a chronic form of EAE) suggests a chronic disorder with some remote resemblance histologically to MS, when EAE is induced in non-human primates it bears a striking resemblance to ADEM and AHLE and not to MS as already discussed. Experimental allergic encephalomyelitis in lower animals, i.e. mice and rats, classically begins in the spinal cord whilst that in monkeys generally involves the neuroaxis. The infiltrating cells are encephalitogenic, Tcells belong to the CD4 subset and are classed as T helper-1 phenotype cells which secrete a number of cytokines including IFN-g and TNF-a and TNF-a. The exquisite sensitivity of these cells and their MHC restriction have all been characterised on several occasions.43 Demyelination in EAE is a highly complex phenomenon and requires not only specific mature T-cells for a variety of other factors producing co-stimulatory signals, the upregulation of adhesion molecules on both the actual T-cells and on the endothelial cells such as ICAM-1 and VCAM-1. Suffice it to say that the immunological reactions involved in EAE, whilst of clear importance in their own right in elucidating pathogenesis, have not been shown to occur in humans. Thus therapeutic strategies based on findings in EAE have failed to succeed in humans.43, 44
Extensive studies have therefore been made of this animal model since all parameters can be examined and controlled. The precise mechanisms of how lymphocytes traffic in the brain in EAE have been elucidated, as has been the role of adhesion molecules and other subcellular elements in controlling and directing cerebral lymphocyte traffic.23 This supposes that the traffic of lymphocytes in the brains of mice is similar to that of lymphocytes (when they do occur) in the brains of patients with MS. Gulcher et al. argued in examining these data: ‘This comment reflects the leap of faith that often characterises the literature on neuroimmunology of multiple sclerosis.’46 Raine accepted that it was impossible to know the dynamic movements over time of cells within the parenchyma of the brain in MS and assumed that such cellular traffic in the animal model, i.e. EAE, was similar to MS.23
Further criticisms can be made for accepting that several of the immunological reactions which occur in animals with EAE and in patients with MS are similar. One of us demonstrated many years ago that, using in vitro techniques, one could detect circulating sensitised lymphocytes in non-human primates immunised to develop EAE, and show that sensitisation occurred to myelin antigens.27, 28 Detailed and repeated identical experiments on patients with MS failed to show a singular case in which clear-cut sensitisation to myelin antigens could be detected.30, 33 However, sensitisation was demonstrated in patients with ADEM and in patients with AHLE.30 These results have been confirmed in different laboratories33 but there has not yet been any confirmed report that such sensitisation occurs truly and specifically in patients with MS as compared to controls and to patients with other neurological diseases.
A large number of potential MS therapies have been envisaged from experiments in animals with EAE. These include vaccination with specific encephalogenic T-cells or peptide fragments derived from similar cells, antibodies against the T-cell receptor, modification of the MHC molecule, anticytokine treatments and the intravenous, intranasal or oral administration of different encephalogenic peptides and proteins. The list is far from complete but it should be stressed that the application of these potential therapeutic techniques to humans has been consumately associated with failure.10
MS AS A POSSIBLE AUTOIMMUNE DISEASE
Multiple sclerosis is one of the most common neurological disorders and certainly the most common human demyelinating disease. It was first described by Carswell from Scotland and Cruveilhier from France in the 1830s, but its exact pathogenesis is still unknown. The classical lesion of MS is the plaque, an area in which there is relative preservation of axons, loss of myelin and digestion of the myelin by macrophages and microglia. Over time the lesion shows pronounced astrocytic gliosis and the final product is a gliotic scar. The plaque is centred round a blood vessel and there may or may not be a mild inflammatory infiltrate of lymphocytes. The vessel involved is usually a small vein, but may be a small artery.
Still no general agreement exists on what precisely happens in the development of this MS plaque. Earlier students such as Lumsden found that the myelin disintegrated in the absence of any infiltrating cells.45 Dawson, however, noted initial pallor of the myelin with an increase of lipid-containing macrophages.34 Most scholars are certain that in some cases there may be no inflammatory cells initially, while even in the developed plaque these are predominantly described as minimal.36 Electron microscopic analysis has shown that myelin breakdown is concomitant with the arrival of the infiltrating macrophages. Indeed, early ultrastructural studies showed changes in the myelin and in oligodendrocytes but without any infiltrating cells being present. The initial hypercellularity in the plaque is due to the presence of astrocytes and infiltrating microglia. Lumsden pointed out that gliofibrillogenesis begins simultaneously with demyelination. The histological features of MS are readily distinguished from other conditions by lack of petechial haemorrhages, accumulated haemosiderin and that of conspicuous oedema.45 It is often stated that the lesions of MS localise in a random fashion but on more careful analysis there is a distinct symmetry when small plaques and all demyelinating activity are taken into account. Lesions of MS usually tend to occur in the cervical cord before involving the cerebral hemispheres. Indeed, the symmetrical distribution of plaques was regarded by Lumsden as one of the most striking statistical findings in that disorder.45
Plaques grow slowly and it has been estimated from statistical studies of patients with long-standing MS that the rate of growth of a plaque is between 2 and 4 mm a year. The demyelinating process however can be more rapid or more severe. It is of extreme interest that Lumsden further argues ‘The basis for this predilection of the multiple sclerosis process for the optic pathways and cord is still, in the writer’s view, the major riddle in the pathology of the disease.’45 As postulated by Oppenheimer,47 these two areas are constantly being traumatised; we move our eyes conjugately in all directions each day and we move our neck laterally and in a flexion/extension arc constantly (whether or not we have cervical spondylosis which would exaggerate the movements). Such movements could predispose to disruption of the blood-brain barrier locally. Brain and Wilkinson noted that cervical spondylosis may be associated with MS and they suggested that ‘The effect of cervical spondylosis upon the spinal cord may be to make it more susceptible to the lesion of multiple sclerosis at that level.’48 They found that in patients with cervical spondylosis and MS at post-mortem there were often widely disseminated plaques of demyelination throughout the brain, particularly in the spinal cord at which site the changes were most conspicuous in the cervical cord segments and most obvious at the levels where spondylosis had occurred.48 This association of MS and cervical spondylosis has been confirmed by Burgerman et al.49
Plaques in the spinal cord are usually longer than those occurring elsewhere. Symmetry of plaque formation is often seen, particularly with bilateral involvement of the fifth nerve entry zone in the pons. In the classic early plaque, special histological stains show preservation of axons with myelin being the primary target. Accompanying the damage to myelin is gliofibrillogenesis with the development of grossly hypertrophied astrocytes. Myelin is broken down into ‘myelin balls’, i.e. droplets of fat, crystallised lipids and granules. The simultaneous vigorous astrocytic response to the breakdown of myelin is possibly the earliest pathological observable abnormality.34 Microglia are not involved until there is myelin breakdown, whereas reactive astrocytes almost appear simultaneously as myelin breakdown. Microglia are activated as a normal secondary event and would be so activated if damage to the myelin were to occur in another way. The remarkable response of astrocytes in MS has been commented on by many authors.34, 45, 51 Other than the response that one finds in prion disease, the astrocytic response in MS is exceptional. Charcot himself commented on this. Simultaneously gliofibrillae appear and there is astrocyte swelling. Such changes in astrocytes bear a relationship to the mechanism for development of a glioma.
The pathological features associated with the initiation and development of acute lesions continue to remain controversial. Lumsden was of the view that demyelination occurred first and, simultaneously, there was astrogliofibrillogenesis and then involvement of microglia and haematogenous cells and, subsequently, invasion by lymphocytes.45 Dawson held similar views.34 He also, in reviewing the earlier literature, found many examples and cases in which on histological examination there was no inflammation whatsoever. Serious scholars of the classical histological lesions of MS have noted scant or even absent ‘inflammatory’ cellular reactions.25, 52 Seitelberger stated that the demyelination of the acute plaque occurred ‘in the absence of mononuclear immune cells.’52 Indeed, as exemplified by several investigators, Guseo and Jellinger found in a detailed study of 19 cases of MS with a short clinical course (the mean of less than one year) a significant number of these patients had no perivascular infiltrate in the CNS.53 The absence of lymphocyte cuffing in patients with acute MS is not the exception and is said to occur in one-third of all cases.36 Lymphocytes are a normal occurrence in the perivascular spaces. Similar infiltrates, in fact exceeding that found in MS, may occur in a wide variety of pathological conditions including trauma, infarcts, tumour and following infections. Most investigators who have looked for lymphocytes in contact with the myelin sheaths and oligodendroglia found that ‘the number of lymphocytes that leave the perivascular compartment and enter the parenchyma is very small, both in absolute numbers and relative to the number of macrophages present.’54–8
There are multiple reports of detailed and sophisticated immunological analysis of the inflammatory cells that are found in the brains of patients with MS. Clearly, it is imperative to compare their type and composition with that found in non-specific conditions with no primary immunological pathogenesis. Similar cells are found in a large number of other conditions including adrenoleukodystrophy,59 a disorder of inborn metabolism, and in neurodegenerative disorders such as amyotrophic lateral sclerosis.60 Similar infiltrates are found up to 40 years later in the spinal cord of those with poliomyelitis.61
Dawson et al.34 were of the opinion that the myelin sheath was first to be affected and simultaneously changes in the neuroglia occurred. There was then alteration in the permeability of the blood vessels. ‘The first cellular increase is secondary to the reabsorptive processes – and infiltration of fat granule cells: this is followed by a proliferation in all the cell elements of the adventitae and, at a later stage, by a modified infiltration of lymphocyte-like cells and a few plasma cells.’34 Since the cause of MS is unknown the significance of the mere finding of a scant haematogenous cellular infiltrate is unknown. As stated, histologists have remarked on the mildness of this infiltrate and Adams was of the view that it was too modest to constitute ‘a florid autoimmune reaction’.62
The finding of inflammatory cells in parts of the brain in patients with MS that contain no myelin such as the retina63 and also the finding of cellular infiltrates away from the plaques64 further raises doubt as to whether such cells have a primary pathological function. Since the earliest lesion in MS is not known, examination of the brains of patients who have had the illness for some time makes it impossible to give a precise age to the inflammatory or demyelinating lesions found. There is still little knowledge as to the precise pathological processes and timing of events that occur in the genesis of the MS plaque. One-third of all MS plaques may not contain cellular infiltrates, and such infiltrates are often scant, lack aggressiveness and indeed may be found in parts of the brain which contain no myelin. Reliance on the presence of these inflammatory cells as the prime pathological process in the disease therefore seems to be totally unfounded. The persistently aggressive nature of the illness classically seen in primary progressive MS but even in the relapsing/remitting type brings the disorder more in line with a neurodegenerative disease than with acquired inflammatory disorder. Indeed Confavreaux and colleagues showed that patients with MS had a progressive course that was not significantly influenced in its progression by relapses and remissions.65 This finding may help to cement what is in our opinion a false separation of primary progressive MS from the other types. Our contention is that there is only one type of the disease with different rates of clinical progression. In our opinion, too, the pathological lesions are identical in both conditions and whilst it is suggested that there may be more inflammatory infiltrates in the secondary progressive disorder, this may be readily explained by the rate of tissue destruction.66 Furthermore, the finding of minor differences in the presence of oligoclonal bands in primary progressive as opposed to the remaining types do not detract from this opinion.66 The presence of oligoclonal bands is non-specific and relates to breakdown of the blood-brain barrier allowing lymphocytes to produce immunoglobulins within the parenchyma of the brain.
Recent detailed studies looking at axonal loss in MS also support our view that MS is a neurodegenerative disease.67 Generalised axonal loss correlates well with clinical deterioration compared with ‘any other measure of structural central nervous system (CNS) change investigated’.67 We will comment later on magnetic resonance spectroscopy studies on total Nacetylaspartate in the CNS white matter but here again there is good correlation with functional impairment.
Immunological findings in MS
The present hypothesis of the pathogenesis of MS is that it is an autoimmune inflammatory condition triggered by an unknown infectious agent; the basis of this hypothesis is that T-cells, specifically sensitised to an unknown antigen, invade the nervous system and cause immunological damage to myelin. A voluminous literature has grown up documenting the innumerable minimal changes found in the peripheral blood and cerebrospinal fluid of patients with MS. No one specific abnormality has ever been found and confirmed.
Attention should be given to the fact that many of these claimed findings are said to occur during the active phase of the disease, i.e. as the patient exhibits new symptoms, whilst comparative studies are done during the quiescent phase. It should be realised that for every new symptom that a patient may develop there may be at least ten demyelinating episodes occurring in the brain visible on a magnetic resonance imaging (MRI) scan, most of them clinically silent. Differentiating activity of the disease merely by documenting exacerbations or new symptoms is clearly open to misinterpretation.68, 69
In an endeavour to support a possible immunopathological basis for MS, researchers have turned to the blood and cerebrospinal fluid to identify any specific immunological abnormalities that correlate with the disease. In animals with EAE it is possible to demonstrate sensitisation of T-cells against components of myelin. Indeed, this phenomenon is also positive in humans with ADEM and AHLE, but has never been confirmed to occur in patients with MS. There are no definitive findings on conventional immunological techniques that polyclonal specific T-cell immune responses against myelin occur in patients with MS.
Using the advanced method of cloning different subsets of T-lymphocytes, it has been claimed that specific clones of T-lymphocytes sensitised to myelin-basic protein are more frequent in patients with MS than in controls. These experiments were repeated, however, and found wanting. 70, 71 Further studies failed to show that there were increased numbers of specific sensitised clones of T lymphocytes to myelin antigens in patients with MS.71–4 Notwithstanding these failures, the protagonists have put forward the idea that the T-receptor gene might be different in T-lymphocyte clones from MS patients as compared to the T-cell receptor gene usage of similar clones in patients without MS. This hypothesis also has been found wanting.75 Other claims regarding the Tlymphocyte and sensitisation to myelin or myelin-specific peptides have been made but have not stood the test of time.76
A wide variety of other immunological abnormalities have been claimed to occur in MS but are non-specific. Among these is the claim that during relapses patients have a decreased suppressor cell function77 and that these cells show a normal function when the patient is asymptomatic and decreased activity during remissions. Again the poor correlation between exacerbations, new symptoms and active demyelination should be noted. This again has been shown to be a non-specific phenomenon since suppressor cell function can vary with a number of abnormalities of the brain, including trauma and brain tumours.78, 79 Indeed, studies of the CD8 suppressor cells in MS demonstrated that they produced normal cellmediated lysis of alloantigens and that immunoglobulin secretion might be increased.80
A variety of other abnormalities involving T-cell subsets and cell-mediated immunity has been described in MS. These have never been consistently confirmed nor proved to play a primary role in pathogenesis; similar abnormalities have been found in patients with other neurological diseases.81 Serial analysis of T-cell activation markers and detailed analysis of cytokines failed to show any reproducible abnormality.82 No firm evidence of an abnormality of T-cell lymphocyte immunity, or of specific sensitisation to myelin antigens or other neural antigens, were confirmed.30, 33 Clearly, the many claims for humoral abnormalities have not stood the test of time nor have the many studies on immunoglobulin abnormalities found in the cerebrospinal fluid.
Attempts at therapy based on experiments in rodents with EAE have so far failed. In example, it has been shown that rats given myelin antigens orally may fail on immunisation with encephalitogenic proteins to develop EAE: oral tolerance has therefore been induced in rats. In a huge trial in humans oral tolerisation to myelin antigens in patients with MS was attempted; no beneficial effect was observed. These experiments were perhaps a further cautionary tale in accepting the theory of autoimmunity in MS.83
Association with autoimmune diseases
In the early 1960s Simpson proposed a theory of autoimmune aetiology for myasthenia gravis.84 Interestingly, this theory was developed predominantly from studying the patients with the disease and their first and second degree relatives with a demonstrably increased association of other autoimmune diseases in all three. For example, he found that 16 of 440 patients with myasthenia gravis concurrently had rheumatoid arthritis. His observations have been amply confirmed in several studies. No such association with autoimmune diseases and correlations with histocompatibility antigens and other markers were found in MS.85 This is borne out by the scant reports of single case occurrences, suggesting that there is no true association.86, 87 Other workers who were keen to highlight such an association have suggested an association with diabetes mellitus88 yet studies looking specifically at this association failed to show the increased incidence of autoimmune disease and patients with MS, particularly diabetes.89 Broadley et al., by the most convoluted arguments, were unable to show any increase in autoimmune diseases in patients with MS when compared with index controls or population data;90 however, they concluded that autoimmune disease was more common in first degree relatives of patients with MS, a finding which they commented on as being consistent with previous larger groups looking especially at this relationship: ‘The finding of an increased familial rate of autoimmune disease without a corresponding increase in patients with multiple sclerosis seems counterintuitive.’90 Lumsden in a careful post-mortem study of a very large number of patients with MS was unable to show any evidence or stigmata of autoimmune disease.35 Broadley et al. found the highest association in the first degree relatives with thyroid disease but this is a difficult and misleading condition to use as a prime example of an autoimmune disease.90 Thyroid disease is extremely common, may be occult and it is difficult to determine its exact incidence. Indeed, a very detailed study from France showed an overall decrease of autoimmune disease in the first degree relatives of patients with MS, whilst reporting a high prevalence of Graves’s disease.91 Clearly no association of autoimmune disease with MS occurs and the absence of any increased incidence in such disorders in the probands leave us in no doubt that no true association exists.
Effect of immunosuppressive/immunomodulationin MS
The importance of the autoimmune hypothesis is well displayed in treatment trials with various and powerful immunosuppressive drugs. The use of these drugs adds another pitfall in the treatment of the condition. The diagnosis of MS depends on a number of different criteria combining clinical and paraclinical factors;92 the criteria which were newly introduced are helpful but still may permit a wrong diagnosis. Even if a correct diagnosis is made, patients still form a heterogeneous study group. Magnetic resonance imaging studies have shown that for each clinical symptom there may be ten silent lesions in the brain. Multiple sclerosis is a complex disease and the natural history of this disorder is not fully appreciated; the mechanisms underlining variable clinical expression are not known, but modern observations suggest that the disease is invariably progressive with a different rate of progression per patient.65, 93 In a carefully conducted study Confavreux and colleagues showed that relapses in essence do not significantly influence the progression of the irreversible disability.65 Other modern observers have noted that ‘The failure of a remission to be complete may be more important than the frequency or severity of antecedent relapses in increasing neurological impairment, and in the transition to a secondary progressive phase.’93 Notwithstanding this, it should also be noted that the majority of patients with MS usually have a rather benign course; even after ten years, 75% of their patients had less than ten lesions on MRI scan of the brain and an extended disability status scale of less than three.94 It can thus be readily seen that there are many difficulties in evaluating patients on a clinical trial.
Clinical trials are, however, in abundance: a rise from 50 such papers in 1965 to more than 300 by the year 2000. This has been described by some as reflecting ‘the sense of excitement in the field of MS therapeutics’,10 but is regarded by us as a sad reflection of the gullibility of researchers in accepting an unproven hypothesis. The authors commented on this sense of excitement philosophically enough to coin the truism: ‘The road to success is paved with failures.’10 The enormous number of trials based on putative immunosuppression and immunoregulatory mechanisms has singularly failed to show a cure or to convey major benefits to MS patients in addition subjecting them to an increased morbidity and mortality.
Some of the trials used (listed in reference 10) were:
1. Immunosuppression: linomide, sulfasalazine, deoxyspergualine, cladribine.
2. Cytokine modulators: lenercept, infliximab, TGF-ß2 (transforming growth factor ß2), IL-10 (Interleukin-10).
3. Inducers of remyelination: IVIg (Gamimune N).
4. Antigen-derived therapies: Oral myelin (Myloral: Al-100), APL (altered peptide ligand: CGP77117; NBI5788), DR2:MBP82-102 (AG284).
5. T-cell-receptor directed therapies: T-cell vaccination, T-cell receptor peptide vaccination.
6. Interferons: (IFNß-1a and IFNß-1b).
7. Glatiramer acetate (copolymer-1).
Claims for the powerful immunosuppressor, cyclophosphamide, led to the widespread use of this drug in MS in Europe and the US.95 The initial studies were not double-blinded, but double-blind, placebo-controlled studies failed to show any improvement.96, 97 Similar results were found with cyclosporine98 and with azathioprine.99
Brain and total lymphoid irradiation and plasma exchange have also been tried. The collected reports of these and other immune-mediated therapies, aimed at ‘Modifying the course of multiple sclerosis’, make for sad reading since a review of the vast number of different trials show, no definite benefit.100 The dramatic failure of trying to induce oral tolerance to myelin-basic protein in humans with MS has even been the subject of a best-selling book.83
ß-interferon and glatiramer in MS
The two leading immunomodulating drugs for which beneficial claims have been made are glatiramer and INFß interferon.10, 101, 102 In a recent editorial data on glatiramer were said to provide reassuring information on its beneficial effects in MS:10 such data as exists with both ßinterferon and glatiramer has to be looked at with extreme caution before total acceptance. Glatiramer ‘is thought to act by inducing a population of regulatory (Th-2 type) T-cells that migrate to inflammatory sites in the central nervous system (CNS), where they are activated by cross-reacting myelin antigens to exert their beneficial “bystander effect”.’10 This process may take several months to develop. Such an explanation defies belief and lies more in the realm of science fiction.
In a study looking at the mechanisms of action of interferons and glatiramer acetate we note that ‘Over the past decade multiple sclerosis patients have benefited enormously from therapeutic research efforts.’101 Sadly we cannot readily accept this statement from our own clinical experience or from the data provided. Others have questioned the efficacy of such trials2 and in looking at the effects of copolymer 1 and ß-interferon found that the ‘copolymer 1 placebo patients fared better than the interferon treatment group’.2 They drew attention to the fact that ‘comparison between studies with even slightly different end-points is extremely difficult.’2 Of extreme importance is the caveat of Steiner and Wirguin that ‘all three studies report a very similar reduction (around 30%) in the relapse rate’.2 They highlight that ‘the almost identical results of clinical trials using different agents, and their inability to go beyond the 33% line, raise the possibility that the entire observed benefit is only a placebo effect, and that the significant deviation from the true placebo might be the outcome of partial unblinding of patients by the side-effects’.2 Similar results were found by Calabresi where improvement with ß1a-interferon was precisely 33%.102
In a recent review of the possible benefits of interferon in relapsing-remitting MS, The Cochrane Review drew attention to 208 articles of which only seven met all the selection criteria and formed the subject of their conclusions.11 The variable quality of the trials, the inadequate methodology, the very high proportion and incomplete description of drop-outs, and the failure to adhere to the strict original intentions of the trial detract seriously from any claims that were made.11 These trials should be considered as single- rather than double-blind. They drew attention to the fact that if interferon-treated patients who had been removed from the study were deemed to have worsened, the significance of the reported effects was therefore lost. The efficacy of interferon on both exacerbations and on progression of the disease was modest after one to two years.11
The precise action of ß-interferon is unknown but an important effect is that it reduces the permeability of the blood-brain barrier, a mechanism that may explain its modest benefit rather than immunomodulation.12 It is said to rapidly block the blood-brain barrier leakage, within two weeks, as seen by gadolinium-enhancement scans.12 Glatiramer acetate also has an effect on the blood-brain barrier but less dramatic resolution of gadolinium-enhanced MRI activity. In a recent issue of Neurology devoted to ‘Practical issues in the management of multiple sclerosis’, a number of investigators look at the action and the clinical results of treatment by glatiramer and interferons. They all begin with a statement that MS is an immunologically mediated disease and all are influenced by the comparison they make with EAE. Dr Dhib-Jalbut wrote of the amplification of immunoreactivity ‘in the central nervous system where T-cells are further activated by antigens presented on microglia resulting in the secretion of pro-inflammatory cytokines and chemokines that attract and retain inflammatory cells in the CNS’.101 We have discussed in this paper the fact that there is no convincing evidence for any of these statements, indeed no antigen has ever been identified that is specific for MS. Dr Dhib-Jalbut further goes on to state that the mechanisms of action in MS of ß-interferon is by reducing T-cell activation, inhibiting interferon-gamma effects, induction of immune deviation and inhibition of the blood-brain barrier. We would humbly suggest that the data surrounding these putative mechanisms would need to be further evaluated, strengthened and confirmed before being accepted.
Glatiramer acetate (Copolymer-1) is a synthetic molecule made of four amino acids: glutamine, leucine, alanine and tyrosine. The same acids are found in myelin-basic protein and because myelin-basic protein has been suspected, but never proved to be, the antigenic component involved in MS, an intellectual jump occurs in assuming that glatiramer acetate, through its action involving immune attack on myelin-basic protein, allows one to use it in the treatment of MS. Again the original work on evaluating copolymer activity was that it blocked the induction of EAE in animals. Glatiramer has been stated to bind to human leukocyte antigen (HLA) Class II (DR) molecules as a result of this possible interaction based on data from its use in animals with EAE, and it is said to inhibit myelinreactive T-cells by inducing anergy in such cells and bringing about anti-inflammatory TH2 cells which have ‘bystander suppression in the CNS’ along with ‘neuroprotection’.101
In the same issue of Neurology Calabresi describes the disease-modifying therapies available for relapsingremitting MS.102 He reported the result of a two-year study of placebo versus ß-interferon-1a. He found the beneficial effect, a reduced exacerbation rate of 34%, whilst in a further study ß-interferon-1a (Avonex) he was able to demonstrate a beneficial effect of 32%.102 This finding was replicated in yet another study that compared placebo with interferon-beta-1a (Rebif).102 Examining the effects of ß-interferon-1b, ß-interferon-1a (Avonex) and Copolymer-1, Steiner and Wirguin noted that the ‘copolymer 1 placebo patients fared better even than the interferon treated group’2 despite the fact that both studies were performed in the same country with patients from an identical population pool. An analysis of these three studies showed that they all had effect but the effect was all around 30% reduction of the relapse rate. These studies have a similar effect on those recently quoted in the US for both glatiramer and ß-interferon.101, 102
In 1955, Beecher published his important paper on ‘The powerful placebo’, where he showed a placebo effect of 30%.103 In a trial of transfer factor in patients with MS one of us was struck by the extraordinary placebo phenomenon that was also observed in patients with MS treated by different techniques.104, 105 We would therefore agree with the recent critiques on reporting clinical trials.106, 107
MS in infants
It is difficult, if not impossible, to induce EAE in neonatal animals;108 immunisation is only successful after a certain age. This is very important since the age at which animals become susceptible to developing EAE corresponds in humans to about the age of two years. This phenomenon of relative resistance to disease is also seen in the histology of ADEM in infants who until the age of two develop an encephalopathy rather than an encephalomyelitis after the appropriate inciting event109 which is characterised by the lack of infiltration of mononuclear cells.109
If sensitised lymphocytes invading the brain are thought to be involved in the pathogenesis of MS, one might therefore not expect to find MS occurring below the age of two. However, there have been several reports of MS occurring in infants.110–13
MS and HIV infection
If MS is an autoimmune disease a particularly informative setting for studying this would be in patients who have MS and are concomitantly infected with HIV infection, a situation which is known to induce a severe state of immune deficiency. Berger et al.114 reported seven such patients, six of whom developed an immunodeficient state after HIV infection but who continued to have relapsing/ remitting MS. Some of these patients had brain biopsies, whilst another died and a full autopsy was performed; there was no doubt about the diagnosis. These data would argue strongly against any autoimmune aetiology, i.e. T-cell mediated autoimmune pathogenesis.
Effect of radiation on MS
When recipient rats involved in passive cellular transfer of EAE were previously irradiated, they were found to have more virulent disease than non-radiated animals. Total body irradiation of these recipient animals did something to them that allowed the passively transferred sensitised cells to induce a more rapid and severe encephalitis.115 In other experiments, however, irradiation of the donor rat reduced the severity of EAE, but specific focal spinal cord irradiation enhanced its development.116 These data are consistent with the known facts that irradiation damages the blood-brain barrier. Damage to the blood-brain barrier certainly enhances EAE. Indeed, in passive cellular transfer experiments, focal damage to one area of the brain three days before transfer will render the animals exquisitely sensitive to developing EAE and they will develop it first within hours in the area of increased blood-brain barrier breakdown.117
Acute radiation therapy, particularly at tumouricidal doses, may disrupt the blood-brain barrier and even cause an encephalopathy.115 Patients with MS who are irradiated would be expected to do badly since disruption of their blood-brain barrier would promote further demyelination. Cerebral demyelinating lesions of MS may appear as single or multiple contrast-enhancing lesions on MRI scans, and be mistaken clinically and radiologically for primary or metastatic brain tumours. Peterson et al.115 reported five such patients who received radiation therapy. Four of the patients received radiation in full tumouricidal doses and had extremely poor clinical outcomes.115 Magnetic resonance imaging scans of patients who have deteriorated after radiation show new lesions which have been verified pathologically as demyelinating. At autopsy in these cases there was profound demyelination.115 Aarli et al. reported a case of a man with MS who had a glioblastoma multiforme and was treated with radiation. He died two months later and the autopsy showed no infiltrating tumour but diffuse demyelinating disease of the white matter.118
Twenty patients with MS received craniospinal radiotherapy and their cerebrospinal fluid IgG level was measured.119 Five patients had a transient decline in IgG synthesis which lasted three to six weeks, ten had totally inconsistent responses whilst the remaining five that were given radiation as well as ACTH and prednisolone, showed a decline in their IgG synthesis rate. What is important to note is that none of these patients was found to have experienced any improvement clinically.119
Total lymphoid irradiation to induce immune suppression126 was also tried in MS: no clinical benefit ensued but a number of deaths occurred, these most likely being secondary to immune suppression,120–23 with an increase in the severity of the disease and the development of infections. The worsening of their demyelinating disease would appear to be the effect of radiation on blood vessels enhancing vascular permeability.115
DISEASES ASSOCIATED WITH MS
Epidemiological studies in certain diseases have shown that there is an increased incidence of related disorders in the probands and first degree and second degree relatives of such patients. Studies applying similar methods to patients with MS have shown that certain diseases other than autoimmune diseases are associated with MS. Classically, these are malignant glioma, including glioblastoma multiforme, neurofibromatosis Type I and hypertrophic peripheral neuropathy.
A compelling series of reports of malignant astrocytomas associated with MS have been published.118, 124–46 Other tumours reported in association with MS have included ependymomas, meningiomas, oligodendrogliomas and odd non-gliomatous tumours but these are very rare and likely to be pure coincidental occurrences.
Multiple sclerosis always occurs first and the tumour generally tends to occur in patients with long-standing disease. In some instances the tumour appears to arise from the adjacent edge of an MS plaque but in the great majority it arises from foci far removed from the demyelinating lesions. It is interesting that there is a very high incidence of multicentric origin.134 In a proportion of MS cases with associated tumours it is impossible to determine whether the tumour is multifocal or not; this is a function of the extent to which the brain is sampled.132 A frequency of 8·75% of multicentricity for all gliomas has been found as compared to 50% when the glioma occurs with MS.134
The number of reported cases almost certainly does not reflect the true incidence of this association. We have personally seen in our institutes a further case of MS, Charcot-Marie-Tooth disease and astrocytoma, and two further cases of the association with malignant astrocytoma. Neurological symptoms occurring in someone with a long-standing disease such as MS are likely to be overlooked and attributed to the original disease. Furthermore, patients with MS who die rarely come to a post-mortem examination. This is important since, in the reported cases of this unique association, several were found at post-mortem examination (that is, the association had in fact been occult).
This association highlights the role of astrocytes in both disorders. It is extraordinary that mitotic figures very rarely, if ever, occur in areas of astrocytic hyperplasia since astrocytes may divide by another mechanism which has been referred to as ‘amitosis’.147 Indeed astrocytes may, like melanoma and other cells, divide under certain conditions by subdivision.148 Glioblastomas, astrocytomas, ependymomas and oligodendrogliomas are all derived from the same progenitor glial cell.
The development of gliomas in patients with MS almost certainly does not occur by chance and is not coincidental. Most astrocytomas are sporadic with no known cause; some, however, are associated with inherited genetic disorders, e.g. neurofibromatosis Type I. This is an autosomal dominant disorder with an incidence of one in 3,000 associated with the development of tumours such as neurofibrosarcoma and optic glioma.149 This condition is also associated with the development of MS so that some intriguing molecular biological deficit appears to link glioma, MS and neurofibromatosis.149–51 That common histogenesis is involved in this association is further supported in the finding of patients with MS, glioma and Charcot-Marie-Tooth disease.133 Furthermore, in patients with both gliomas and MS there is often multicentric origins of the neoplasm which, being a rare occurrence by itself, may suggest a common pathogenesis.
One might have expected oligodendroglia to proliferate in putative disorders associated with MS and the vast majority of neoplasms are either glioblastomas and astrocytomas (Table 1). Similarly, there is no evidence in most cases of a continuous transformation of the reactive glial cells of an MS plaque to an adjacent tumour, thus the neoplastic transformation from reactive glial cells of the MS plaque is unlikely to be the mechanism. Some authors have, however, claimed that the neoplastic transformation of astrocytes has occurred from the activated cells within the MS plaque.141 Malmgren et al. commented on their case in which neoplastic astrocytes were found within the MS plaque suggesting the neoplastic change had arisen de novo within the plaque.141
Ho and Wolfe139 in their study of 20 cases found six astrocytomas that were multiple, which is a far greater number than is usually described as occurring in patients with glioma alone. Currie and Urich stressed that the high frequency of multiple sites of origin when glioma occurs with MS exceeded even ‘the exceptionally high estimate of 20% which Scherer, 1940,124 gave as the frequency of multiple tumours in all cases of glioma’.136 This point is reinforced by Reagan and Freiman, who refer to the very high incidence – up to 50% – of multifocal lesions in patients who have a glioma and MS.134
2. Hypertrophic peripheral neuropathies
Clinical, electrophysiological, morphological and pathological studies have shown abnormalities in the peripheral nervous system in patients with MS. Pollock et al. found peripheral nerve abnormalities in 80% or more with a reduction in myelin thickness.152 One difference was that the demyelinated and re-myelinated segments of the peripheral nerve did not extend laterally as occurred in MS plaques. There was no correlation with clinical findings. Earlier workers considered that these peripheral lesions were associated with malnutrition and avitaminosis.153 Others have attributed them to an autoimmune mechanism; in some cases molecular genetic testing revealed myelin-protein gene duplication, characteristics of Type Ia Charcot-Marie-Tooth disease.154 Interestingly, the molecular abnormalities in Charcot- Marie-Tooth neuropathy are found in intrinsic Schwann cell proteins and not in the axons.155
The data showing involvement of the peripheral nerve system in patients with MS are overwhelming and include altered supernormality in MS peripheral nerve,156 reduced myelin thickness of internodes,152 ultrastructural abnormalities in Schwann cells,157 gross onion bulb neuropathy,158–60 normal regional curare test,161 abnormal single fibre EMG,162 prolonged refractoriness of sensory nerves163 and abnormal slowing of motor conduction as evidenced by collision technique.164 In a classic case of severe onion bulb neuropathy associated with MS,158 both of these diseases began simultaneously: whatever caused MS was likely to be involved in the initiation of onion bulb neuropathy.158
It would be reasonable to consider a promoting factor that caused proliferation both of Schwann cells and glia since the rise in the CSF protein early in the course of the disease suggests root involvement at the time of developing MS. Schwann cells and glia must therefore share a common receptor. The unique finding, in this case, of Schwann cells within the plaque in the spinal cord is very remarkable. Some workers have reported that peripheral myelin can occur in MS plaques.165, 166 These earlier workers suggested that there must be pluripotential stem cells within the cord so that the plaque contains in these unique cases multiplying glia and Schwann cells that arise from such pluripotential cells. The rapid proliferation, in some way, perhaps by diverting normal metabolism may cause demyelination. For such an event to occur the milieu in the spinal cord must contain such a proliferating factor(s). Whether this comes from the cord itself or from the blood is unknown. Neuregulin and the erb B receptors are likely candidates.167
We were intrigued to find a young pregnant woman who presented with classical neurofibromatosis Type I and a progressive paraparesis. Investigations revealed the paraparesis to be secondary to a spinal cord MS plaque with multiple lesions throughout the white matter of the CNS. Two other cases referred to us suggested this association might be more than coincidence and a review of the literature showed neurofibromatosis and MS were reported to occur together.149–51, 168, 169
Neurofibromatosis is an autosomal dominant condition with a wide intrafamilial
variability in clinical manifestations. About 50% of all cases represent
new mutations. There is an association with glial nodules in the brain
and spinal cord and an increased incidence of glioma. The NF1 gene consists
of 350 kb of genomic DNA and encodes for a protein of 2,818 amino acids,
i.e. neurofibromin which is expressed in many different tissues. Its precise
role is unknown but it does act as a GTPase-activating protein in the same
pathway of signal transduction as ras6 and it may act as a tumour suppressor
in controlling the proliferation and differentiation of cells. Indeed,
for normal embryological development to occur the spatial and temporal
expression of the NF1 gene is crucial.6 The gene is located
on chromosome 17q11·2.170 These genes are enclosed in
an intron of this gene which includes oligodendrocyte myelin glycoprotein.
This membrane glycoprotein appears in the human brain around the time of
myelination.6 The finding of an increased association of NF1
with glioma170 and MS150, 151 suggests that this
may reflect a shared pathogenesis. The finding of the gene for NF1 on chromosome
17q11·2 is of particular interest and importance.6
MS, A METABOLICALLY DETERMINED NEURODEGENERATIVE DISORDER?
Evidence from neuroimaging
Since the earlier work of Charcot, Dejerine, Marie and Dawson, neuronal loss in MS has been recognised, and more recently, this has been confirmed by MRI, ultrastructural and magnetic resonance spectroscopy (MRS) studies.171 Neuronal loss in MS is an essential part of the disease since demyelination alone does not adequately explain the functional impairment and longterm disability.172 It is also important to stress here that there is significant heterogeneity in the pathological pattern of MS demyelination and myelin loss in MS is likely to be the final pathway of multiple pathogenetic mechanisms.173
Data from MRS clearly point to early metabolic changes in evolving MS lesions. Based on new magnetic resonance technology, it is now clear that metabolic changes appear first, often weeks before (e.g. three months in one study) the appearance of gadolinium (Gd) enhancement in MS.174 Total brain (rather than regional) N-acetyl aspartate (NAA), a marker of functional neuronal mass, correlates well with relapses.175 Reduction of total brain NAA is an excellent predictor of focal relapses in MS, suggesting that relapses (and their biological marker, demyelination) are related to global reduction of brain metabolic function (reduced NAA), reversible in some cases.172 Since white matter has a limited metabolic repertoire, compromised areas of white matter will retain water and swell. Increased water content in the metabolically compromised white matter will appear as an increased signal in the T2- weighted images in MRI. Focal breakdown of blood-brain barrier (lesion enhancement) probably occurs in response to the metabolic changes, allowing macrophages to be drawn into the lesion. These cells will phagocytose the swollen myelin, representing the initial ‘inflammatory’ infiltrate well documented in Dawson’s original observations.54 That global metabolic compromise is an integral component of developing MS lesions is indicated by the fact that grey matter lesions are always present, notably in the basal ganglia and at the grey-white junctional areas of the cortex.175 This is distinct from primary oligodendroglial disease, as seen in progressive multifocal leukoencephalopathy (PML) that spares grey matter and (unlike MS) involves the subcortical ‘U’ fibres.
Unlike conventional MRI, magnetic resonance spectroscopy studies in MS have shown very significant axonal damage, even in the normal appearing white matter (NAWM)176, 177 early in the disease.174 Magnetic resonance spectroscopy measures of NAA correlate with clinical changes in MS disability. Multiple sclerosis patients with progressive cerebellar deficit and low cerebellar functional scores have lower cerebellar NAA compared to MS patients with fewer cerebellar symptoms and the reduction in their NAA.178 N-acetyl aspartate measures of corpus callosum in MS showed a progressive decline in the NAA resonance in serial study.179 This is also true for the spinal cord where reduced NAA levels correlated with reduced axonal areas in the spinal cord cross-sections in an autopsy study.180 In contrast, post-mortem tissue sampling by using MRI has confirmed low specificity of T2-weighted imaging. Using in vivo quantitative magnetic resonance studies, T1-weighted images were found to be more relevant than T2-weighted images in identifying lesions that cause disability. T1 hypointensity (‘black holes’), on the other hand, strongly correlated with axonal loss.181
Metabolic changes were observed well before the appearance of magnetic resonance lesions in serial MR studies that included proton MR spectroscopic imaging (MRSI), contrast-enhanced MRI imaging and volumetric lesion studies of 25 patients with mild to moderate clinical deficits over a period of two years.174 Regional metabolite changes were both dynamic and reversible. In four patients strong lipid peaks in the absence of Gdenhancement and magnetic resonance-defined lesions were observed, clearly indicating that demyelination can occur independent of ‘inflammatory’ changes. It should be remembered that, when examined at autopsy, onethird of all plaques contain no inflammatory infiltrates.64 While focal reductions in NAA levels did not always imply axonal loss, reduction in whole brain NAA was a putitive marker of the MS disease burden.174 As compared to healthy controls, reduction in whole brain NAA in this study was found to be greater in older rather than younger patients consistent with the clinical experience that older MS patients usually have worse outcomes. The agedependent decrease of whole brain NAA in MRS suggests that progressive neuronal cell loss is a cardinal feature of the disease, very similar to other neurodegenerative diseases. The similarity of cerebellar MRS changes in MS patients with cerebellar symptoms and autosomal dominant cerebellar ataxia attest to this view.178
Direct pathological studies of the NAWM have revealed axonal loss and the degree of volume loss was greater than that from the anticipated loss from MS lesions alone.177 Recently, it was shown with T1-weighted magnetic resonance follow-up studies that the degree of ‘inflammatory activity’ is a poor predictor of not only T1- but also T2-lesion load at long-term follow-up, indicating that the T2-lesion load is not an accurate measure of the MS pathology.182 Consistent with the previous observations, the authors of the latter study found that the baseline T1-lesion load was the single most important factor in the subsequent increases in hypointense T1-lesion load. Taken together, these findings clearly confirm that the so-called inflammatorydemyelinating indices in conventional MRI (abnormal increased signals in T2-weighted MRI and Gdenhancement) do not predict long-term MS disability and are poor outcome measures for treatment trials designed to prevent progression to disability. It also appears that long-term MS disability is largely predetermined by the axonal loss already present at the symptom onset, a feature characteristic of neurometabolic and degenerative diseases. Imaging and pathological studies in chronic demyelinated cervical spinal cord plaques clearly indicate that slow axonal degeneration rather than acute change is responsible for chronic disability in MS. A predominance of widespread NAWM changes over focal lesions appears to be the hallmark of primary progressive MS.172 These observations support the conclusions of a recent MS epidemiologic study proposing a biological dissociation between relapses and progressive disability being characteristic of the natural history in MS.65
Relationship of glial proliferation and metabolic changes to MRI abnormalities
Diffusion of water molecules in relation to the directional organisation of the myelinated white matter can be measured by diffusion tensor MRI. In this form of neuroimaging, magnitude and translational motion of the water molecules (‘anisotropy’) with respect to the white matter fibre tracts can be defined. Myelin and cell membranes provide natural barriers to diffusion across the white matter fibres.183 As a result, the water molecules can diffuse only along these fibres. Therefore, a high degree of anisotropy is expected in normal myelinated white matter fibres and this has been confirmed in histologic and imaging studies.183–5 On the other hand, decreases in diffusion anisotropy have been shown to occur in diseases that affect myelin or axonal integrity, such as MS, neurodegenerative diseases, cerebral ischaemia and leukodystrophies.185–9
In MS, reduction in the functional anisotropy in diffusion tensor MRI precedes any other change in the conventional MRI. This applies to patients with clinically definite MS and normal T2-weighted magnetic resonance scans190 and patients with clinically isolated syndromes suggestive of MS.191 The anisotropy measurements also correlate well with evidence from histologic and MRS studies.192 It is thus clear that the disease process in MS extends well beyond the borders of the plaque identified in the T2- weighted images. There is a progressive gradient in the decline of anisotropy observed in MS from the NAWM to the peri-plaque and intra-plaque white matter with the latter showing the most extensive changes. A progressive decrease of magnetisation transfer ratio is also seen to extend from the remote NAWM towards the plaque.192 In contrast, the white matter abnormalities in ADEM do not extend beyond the focal areas of injury and the magnetisation transfer ratios of the uninvolved brain and spinal cord in ADEM are practically identical to the values observed in the healthy control subjects.193 The anisotropic changes in the normal appearing brain tissue is always present in all clinical phenotypes of MS irrespective of their presentations.190, 191, 194
The basis of the anisotropic gradient that extends from the NAWM to the fully formed MS plaque in MS is important in the understanding of its pathogenesis. Irrespective of the clinical phenotype, two consistent abnormalities are revealed in the MS brain by the newer imaging technologies. Firstly, there is reduced brain metabolism and secondly, the water diffusibility is reduced along the white matter fibre tract, and the plaque is centred round the area where the anisotropy is lowest. Such findings are unique to MS, being absent in ADEM, Devic’s neuromyelitis and in patients with other systemic immunological diseases associated with multiple T2- weighted signal abnormalities in the MRI.195 Changes in MRS and diffusion tensor imaging appear to occur before other magnetic resonance changes and correlate with functional impairment in MS.192 Results of a follow-up study showed that 80% of the newly formed MS lesions are hypointense on unenhanced T1-weighted scans associated with the highest anisotropy changes.196 However, more than half (44%) of these new lesions returned to isointensity spontaneously during the course of the follow-up.196 This spontaneous reversibility is very characteristic of metabolic disease. Data from the MRS and diffusion tensor imaging do, therefore, suggest that MS is a neurodegenerative disease in which the rate of myelin loss is metabolically determined. In this paradigm, plaque formation is a subacute process that follows a failure of metabolic reversibility and characteristically occurs in an area farthest from the NAWM. This is comparable to the zonal architecture proposed in the functional anatomy of the hepatic lobule where the metabolic changes tend to be maximum towards the periphery of the lobule.
It may not be out of place to consider possible explanations for reduced anisotropy in MS. As already noted, anisotropic changes in the MS brain are extensive, being abnormal in virtually all white matter regions and always extending well beyond the focal areas of myelin loss.192 In the developing brain, increased permeability and anisotropic diffusion across myelin has been shown to occur before the onset of myelination. Premyelination anisotropy may be due to a number of changes, including an increase in fibre diameter and axonal membrane changes.197 This may explain why demyelination may occur in infancy or in the structural disorders of axons and myelin (leukodystrophies). It has also been postulated that as a result of change in energy metabolism, oligodendrocytes may fail to meet its required energy demand in mitochondrial encephalopathies leading to demyelination. However, anisotropy may also be reduced by glial proliferation.198 Because glial cells are fairly amorphous and glial proliferation is not a structurally organised process, anisotropy will be reduced in areas where glial proliferation occurs.199 It is our contention that the reduced anisotropy in the MS brain is a direct function of glial proliferation and consequent metabolic changes in the myelin lead to plaque formation by way of increased permeability. It is the generalised glial proliferation in MS that accounts for the globally reduced anisotropy; the metabolic changes contribute to the formation of plaque in the areas of maximum compromise.
A final comment has to be made about the so-called ‘inflammatory’ magnetic resonance lesions. The conventional MRI data (T2-weighted and Gdenhancement) in MS merely show that some MS plaques are swollen (oedematous) with a breakdown of the bloodbrain barrier. These changes would certainly be expected if myelin and cell membrane permeabilities are altered. However, to suggest that these magnetic resonance changes in the MS plaques reflect specific T-cell mediated autoimmune inflammatory process has not yet been substantiated. As previously stated, at least one-third of MS plaques in pathological studies show no evidence of inflammatory infiltrate, and the mononuclear infiltrate in the other plaques is either mild or inconsistent. Comparable infiltrates are encountered, as described, in diseased brain due to stroke, tumour or indeed, even in Alzheimer’s or motor neurone disease. Logic therefore dictates that cellular infiltrates are unlikely to be responsible for oedema in the MS plaques or focal breakdown in the blood-brain barrier. This is certainly supported by the data from the magnetisation transfer and diffusion tensor MRI in MS.
Pregnancy and MS
The effect of pregnancy on MS provides an important clue to the nature of this yet unknown factor stimulating glial proliferation and reducing myelin metabolism. Pregnancy has an apparent stabilising effect on the clinical course of MS. Indeed, Confavreux has commented that the effect of pregnancy on MS is better than the treatment effects of ß-interferon or glatiramer.200 However, the threemonth, post-partum period shows an increasing relapse rate. The effect of pregnancy on MS contrasts sharply with systemic lupus erythematosus (SLE), a classical autoimmune disease, where a flare-up of disease is common during pregnancy.
Comparative epidemiology of MS and neurodegenerative diseases
In this review, we have suggested that MS is not an autoimmune disease due to its intrinsic clinical, immunological, radiological and histological differences with ADEM and its experimental model, EAE. In our opinion, MS has the characteristics of a metabolically determined neurodegenerative disorder with strong genetic influence. There is now evidence from epidemiological studies that other neurodegenerative diseases (Parkinson’s disease and motor neurone disease), once thought to be evenly spread and to occur at a uniform rate throughout the world, are influenced by geographic factors similar to MS.201 This analogy can be extended further in that we, and others, have shown that MS may be precipitated, but not caused, by specific trauma to the CNS.50, 202 Trauma has also been implicated in the precipitation both of Parkinson’s disease and of motor neurone disease.203, 204 Of great interest is the finding that electrical injuries are statistically significant in a prospective trial of looking at the role of trauma in MS,205 and both motor neurone disease and Parkinson’s disease have been described as occurring following electrical injuries.206, 207 Further data indicating that MS is a progressive neurodegenerative disorder is found in a recent study looking at clinical analysis of 250 families in which a cohort of 262 pairs of co-affected siblings were studied.208 The authors of this report found that once the disease was established, concordance was more related to the ultimate clinical course than to the number of attacks or presentation and the end result of disability and handicap scores was similar.
The proposition that MS is a prototype autoimmune disease is weak and open to question: MS is not a model of EAE or, indeed, of any known autoimmune disease. Its histology compares more favourably with other forms of demyelination known to be metabolic in aetiology, such as leukoencephalopathies due to mitochondrial encephalopathy, central pontine myelinolysis and Marchiafava-Bignami disease. Many scholars of MS in the past have argued strongly that the disease is toxic/ metabolic in origin.209 The suggested association of a HLA in MS is neither strong nor persuasive. Such data for a claimed association with DR2 would argue against any link with diabetes, since the presence of DR2 protects against this disorder. Whatever immunologic finding that has been claimed to be specific for MS is soon found wanting. There is never a shortage of candidates such as the heat-shock protein or ß-crystalline that is found in brains with other neurological diseases as well as normals.210 It genuinely is time to seek a paradigm shift in the pathogenesis of MS and look anew at the known facts.
Multiple sclerosis remains a disease of unknown aetiology. In recent years, most researchers have uncritically accepted the hypothesis that it is an autoimmune disorder. An in-depth review of the literature failed to support this concept, and the immunological claims for this disease are tenuous and fragile. There is no one specific immunological abnormality found in MS that does not occur in patients with other diseases or in normal controls. The acceptance of EAE as a model for MS is an unfortunate error that has its basis on faith rather than science. Whilst EAE is a good example of an experimental organ-specific autoimmune disorder in animals, it cannot be accepted as a model for MS for a wide variety of reasons. This is particularly important in relation to the development of MS pharmacotherapy. We have analysed the literature on immune-modifying therapy in MS and it is clear that none of these agents can qualify as a candidate therapy under scrutiny.
A clear association of MS exists with other glial disorders of proliferation and differentiation. Based on comparative neuropathology and neuroimaging data, it is proposed that the initial lesion in MS is one of astrocytic proliferation and plaque formation and is a consequence of metabolic abnormality affecting myelin permeability. We believe these pathogenic changes in MS are influenced by a combination of genetic and environmental factors. One of the genes most likely to be involved in MS pathogenesis may be located on chromosome 17. The gene expression in MS is influenced by external factors, and whilst such factors have not been identified with certainty, one of these is likely to be the influence of sunlight, perhaps mediated through vitamin D metabolism.210
This work was supported by the Barclay Trust at the University of Glasgow.
1 Noseworthy JH. Progress in determining the causes and treatment of multiple sclerosis. Nature 1999; 399:A40– 7.
2 Steiner I and Wirguin I. Multiple sclerosis – in need of a critical reappraisal. Med Hypotheses 2000; 54(1):99–106.
3 Bell JI, Lathrop GM. Multiple loci for multiple sclerosis. Nat Genet 1996; 13:377–8.
4 Haines JL, Terwedow HA, Burgess K et al. Linkage of the MHC to familial multiple sclerosis suggests genetic heterogeneity. Hum Mol Genet 1998; 7:1229–34.
5 Kalman B, Lublin FD. The genetics of multiple sclerosis. A Review. Biomed Pharmacother 1999; 53(8):358–70.
6 von Deimling A, Krone W, Menon AG. Neurofibromatosis type 1: pathology, clinical features and molecular genetics. Brain Pathol 1995; 5:153–62.
7 Compston A, Coles A. Multiple Sclerosis. Lancet 2002; 359:1221–31.
8 Johnson MR, Ferner RE, Bobrow M et al. Detailed analysis of the oligodendrocyte myelin glycoprotein gene in four patients with neurofibromatosis 1 and primary progressive multiple sclerosis. J Neurol Neurosurg Psychiatry 2000; 68:643–6.
9 Wrabetz, Feltri, Hanemann et al. The molecular genetics of hereditary demyelinating neuropathies. In: Glial Cell Development – Basic Principles and Clinical Relevance. Eds. Jessen KR and Richardson WD. Oxford: Oxford University Press; 2001; 331–54.
10 Hohlfeld R, Wiendl H. The ups and downs of multiple sclerosis therapeutics. Ann Neurol 2001; 49(3):281–4.
11 Rice GPA, Incorvaia B, Munari L et al. Interferon in relapsingremitting multiple sclerosis (Cochrane Review). In: The Cochrane Library 1. Oxford: Update Software; 2002.
12 Stone LA, Frank JA, Albert PS et al. The effect of interferonbeta on blood-brain barrier disruptions demonstrated by contrast-enhanced magnetic resonance imaging in relapsingremitting multiple sclerosis. Ann Neurol 1995; 37:611–19.
13 Glanzmann E. Die Nervosen Komplicationen der Varizellen der Variola und Vakzine. Schweiz Med Wochenschr 1927; 57:145–54.
14 Remlinger P. Les paralysies du traitement antirabique. Ann Inst Pasteur 1928; 42(Suppl. 71).
15 Aujeszky A. Ueber Immunizierung gegen Wut mit normaler Nervensubstanz. Centralbl Bakt. Jena 1900; 27:5–10.
16 Bassoe P, Grinker RR. Human rabies and rabies vaccine encephalomyelitis: A clinicopathological study. Arch Neurol Psychiat 1930; 23:1138–60.
17 Schwentker FF, Rivers TM. The antibody response of rabbits to injections of emulsions and extracts of homologous brain. J Exp Med 1934; 60:559–74.
18 Rivers TM, Schwentker FF. Encephalomyelitis accompanied by myelin destruction experimentally produced in monkeys. J Exp Med 1935; 61:689–702.
19 Kabat EA, Solf A, Bezer AE. Rapid production of acute disseminated encephalomyelitis in rhesus monkeys by injection of brain tissue with adjuvants. Science 1946; 104:362–3.
20 Freund J, Stern ER, Pisani TM. Isoallergic encephalomyelitis and radiculitis in guinea pigs after one injection of brain and mycobacteria in water-in-oil emulsion. J Immunol 1947; 57:179–94.
21 Adams RD. Chapter 5: A comparison of the morphology of the human demyelinating diseases and experimental ‘allergic encephalomyelitis’. In: ‘Allergic’ Encephalomyelitis. Eds. Kies MW, Alvord EC. Washington: Charles C Thomas;183–209.
22 Levine S. Relationship of experimental allergic encephalomyelitis to human disease. In: Immunological Disorders of the Nervous System. Ed. Rowland LP. Baltimore, Maryland:Williams & Wilkins. Res Publs Ass Nerv Ment Dist 1971; 49:33–49.
23 Raine CS. The Dale E McFarlin memorial lecture: the immunology of the multiple sclerosis lesion. Ann Neurol 1994; 36:S61–72.
24 Behan PO, Currie S. Chapter 4: Acute disseminated encephalomyelitis. In: Clinical Neuroimmunology. London, Philadelphia and Toronto: Saunders Company; 1978; 49– 61.
25 Graham DI, Behan PO, More IAR. Brain damage complicating septic shock. J Neurol Neurosurg Psychiat 1979; 42:19–28.
26 Russell DS. The neurological unity of acute haemorrhagic leukoencephalitis and acute disseminated encephalomyelitis. Brain 1955; 78:369–76.
27 Behan PO, Behan WMH, Feldman RG et al. Cell-mediated hypersensitivity to neural antigens. Arch Neurol 1972; 27:145–52.
28 Behan PO, Kies MW, Lisak RP et al. Immunologic mechanisms in experimental encephalomyelitis in nonhuman primates. Arch Neurol 1973; 29:4–9.
29 McNair Scott TF. Post-infectious and vaccinial encephalitis. In: Medical Clinics of North America. Ed. Sweeney Jr, FJ. Philadelphia and London: W B Saunders; 1967; 701–17. 30 Behan PO, Geshwind N, Lamarche JB et al. Delayed hypersensitivity to encephalitogenic protein in disseminated encephalitis. Lancet 1968; ii:1009–12.
31 Cohen JA, Lisak RP. Acute disseminated encephalomyelitis. In: Eds. Aarli JA, Behan WMH, Behan PO. Clinical Neuroimmunology. Oxford: Blackwell Scientific Publications; 1987; 15, 192–213.
32 Lamarche JB, Behan PO, Segarra JM et al. Recurrent acute necrotising haemorrhagic encephalopathy. Acta Neuropath 1972; 22:79–87.
33 Johnson DA, Hafler DA, Fallis RJ et al. Cell-mediated immunity to myelin-associated glycoprotein, proteo-lipid protein, and myelin basic protein in multiple sclerosis. J Neuroimmunol 1986; 13:99–108.
34 Dawson J. The Histology of Disseminated Sclerosis. Trans R Soc Edinburgh 1916; 50:517–40.
35 Lumsden CE. Pathology of Multiple Sclerosis and Allied Demyelinating Diseases. In: Eds. McAlpine D, Compston ND, Lumsden CE. Multiple Sclerosis. Edinburgh: Livingstone; 1955; 208–93.
36 Adams CWM. Pathology of multiple sclerosis: progression of the lesion. Br Med Bull 1977; 3:15–20.
37 Popovich PG, Stokes BT, Whitacre CC. Concept of autoimmunity following spinal cord injury: possible roles for T lymphocytes in the traumatised central nervous system. J Neurosci Res1996; 45:349–63.
38 Paterson PY. Chapter 1: Neuroimmunology: an overview and personal perspective. In: Eds. Aarli JA, Behan WMH, Behan PO. Clinical Immunology. Oxford, London and Boston: Blackwell Scientific Publications; 1987; 3–25.
39 Aihara N, Tanno H, Hall JJ et al. Immunocytochemical localisation of immunoglobulins in the rat brain: relationship to the blood-brain barrier. J Comp Neurol 1994; 342:481–96.
40 Noble LJ, Wrathall JR. Distribution and time course of protein extravasation in the rat spinal cord after contusive injury. Brain 1989; Res 482:57–66.
41 Fritz RB, Skeen MJ, Jen-Chou CH et al. Major histocompatibility complex-linked control of the murine immune response to myelin basic protein. J Immunol 1985; 134:2328–32.
42 Baker D, Rosenwasser OA, O’Neil JK et al. Genetic analysis of experimental allergic encephalomyelitis in mice. J Immunol 1995; 155:4046–51.
43 Zamvil SS, Steinman L. The T lymphocyte in experimental allergic encephalomyelitis. Annu Rev Immunol 1990; 8:579– 621.
44 Martin R, McFarland HF. Immunology of multiple sclerosis and experimental allergic encephalomyelitis. In: Eds. Raine CS, McFarland HF, Tourtellotte WW. Multiple Sclerosis. London: Chapman & Hall Medical; 1997; 221–39.
45 Lumsden C. The neuropathology of multiple sclerosis. In: Eds. Vinken PJ, Bruyn GW. Handbook of Clinical Neurology. Vol. 9. Multiple sclerosis and other demyelinating diseases. Amsterdam: Elsevier; 1970; 217–309.
46 Gulcher JR, Vartanian T, Stefansson K. Is multiple sclerosis an autoimmune disease? Clin Neurosci 1994; 2:246–52.
47 Oppenheimer DR. The cervical spine in multiple sclerosis. Neuropathol Appl Neuobiol 1978; 4:151–62.
48 Brain W, Wilkinson M. The association of cervical spondylosis and disseminated sclerosis. Brain 1957; 80:456–78.
49 Burgerman R, Rigaminto E, Randle JM et al. The association of cervical spondylosis and multiple sclerosis. Surg Neurol 1992; 38:265–70.
50 Chaudhuri A, Behan PO. Acute cervical hyperextensionhyperflexion injury may precipitate and/or exacerbate symptomatic multiple sclerosis. Eur J Neurol 2001; 8:659– 64.
51 Arstila AU, Riekkinen PJ, Rinne UK et al. Studies of the pathogenesis of multiple sclerosis. Participation of lysosomes in demyelination in the CNS white matter. Europ Neurol 1973; 9:1–21.
52 Seitelberger F. Histochemistry of demyelinating diseases proper including allergic encephalomyelitis and Perlizaeus- Merzbacher’s disease. In: Ed. Cummings NJ. Modern Scientific Aspects of Neurology. London: Edward Arnold; 1960; 146–87.
53 Guseo A, Jellinger K. The significance of perivascular infiltrations in multiple sclerosis. J Neurol 1975; 211:51– 60.
54 Prineas JW. The neuropathology of multiple sclerosis. In: Ed. Koetsier JC. Handbook of Clinical Neurology. Amsterdam: Elsevier Science Publishers B.V.; 1985; 3(47):213–57.
55 Sluga E. Beitrag zur Feinstruktur der Lasionen bei der multiplen Sklerose des Menschen. Wein Z Nervenheilkd Grenzgeb 1969; (Suppl. 2):59–69.
56 Prineas J. Pathology of the early lesion in multiple sclerosis. Hum Pathol 1975; 6:531–54.
57 Tanaka R, Iwasaki Y, Koprowski H. Ultrastructural studies of perivascular cuffing cells in multiple sclerosis brain. Am J Pathol 1975; 81(3):467–78.
58 Prineas JW, Kwon EE, Sternberger NH et al. The distribution of myelin-associated glycoprotein and myelin basic protein in actively demyelinating multiple sclerosis lesions. J Neuroimmunol 1984; 6:251–64.
59 McGuinness MC, Smith KD. Cerebral inflammation in Xlinked adrenoleukodystrophy (Review). Arch Immun Ther Exp (Warsz) 1999; 47(5):281–7.
60 Engelhardt JI, Tajti J, Appel SH. Lymphocytic infiltrates in the spinal cord in amyotrophic lateral sclerosis. Arch Neurol 1993; 50:30–6.
61 Pezeshkpour GH, Dalakas MC. Long-term changes in the spinal cords of patients with old poliomyelitis. Arch Neurol 1988; 45:505–08.
62 Adams CWM, Poston RN, Buk SJ et al. Inflammatory vasculitis in multiple sclerosis. J Neurol Sci 1985; 69:269–83.
63 Lightman S, McDonald WI, Bird AC et al. Retinal venous sheathing in optic neuritis. Its significance for the pathogenesis of multiple sclerosis. Brain 1987; 110:405– 14.
64 Adams CWM. Histology and cellular features of multiple sclerosis. In: A Colour Atlas of Multiple Sclerosis and other myelin disorders. London: Wolfe Medical Pubs Ltd; 1989; 130–9.
65 Confavreux C, Vukusic S, Moreau T et al. Relapses and progression of disability in multiple sclerosis. N Eng J Med 2000; 343:1430–8.
66 Revesz T, Kidd D, Thompson AJ et al. Comparison of the pathology of primary and secondary progression of multiple sclerosis. Brain 1994; 117:759–65.
67 Ganter P, Prince C, Esiri MM. Spinal cord axonal loss in multiple sclerosis: a post-mortem study. Neuropathol Appl Neurobiol 1999; 25:459–67.
68 Isaac C, Li DKB, Genton M et al. Multiple sclerosis: A serial study using MRI in relapsing patients. Neurology 1998; 38:1511–15.
69 Truyen L, Gheuens J, Parizel PM et al. Long-term followup of multiple sclerosis by standardised, non-contrastenhanced magnetic resonance imaging. J Neurol Sci 1991; 106:35–40.
70 Brinkman CJJ, Nillesen WM, Hommes OR et al. Cellmediated immunity in multiple sclerosis as determined by sensitivity of different lymphocyte populations to various brain tissue antigens. Ann Neurol 1982; 11:450–5.
71 Burns J, Rosenweig A, Zweiman B et al. Isolation of myelin basic protein-reactive T-cell lines from normal human blood. Cell Immunol 1983; 81:435–40.
72 Hafler DA, Buchsbaum M, Johnson D et al. Phenotypic and functional analysis of T-cells cloned directly from the blood and cerebrospinal fluid of patients with multiple sclerosis. Ann Neurol 1985; 18:451–8.
73 Tournier-Lasserve E, Hashim GA, Bach MA. Human T-cell response to myelin basic protein in multiple sclerosis patients and healthy subjects. J Neurosci Res 1988; 19:149– 56.
74 Richert JR, McFarlin DE, Rose JW et al. Expansion of antigen-specific T-cells from cerebrospinal fluid of patients with multiple sclerosis. J Neuroimmunol 1983; 5:317–24.
75 Ben-Nun A, Liblau RS, Cohen L et al. Restricted T-cell receptor gene usage by myelin basic protein-specific Tcell clones in multiple sclerosis. Predominant genes vary in individuals. Proc Natl Acad Sci USA 1991; 88:2466–70.
76 Martin R, Voskuhl R, Flerlage M et al. Myelin basic proteinspecific T-Cell responses in identical twins discordant or concordant for multiple sclerosis. Ann Neurol 1993; 34:524–35.
77 Antel JP, Arnason BGW, Medof ME. Suppressor cell function in multiple sclerosis; correlation with clinical disease activity. Ann Neurol 1979; 5:338–49.
78 Quattrocchi KB, Frank EH, Miller CE et al. Suppression of cellular immune activity following severe head injury. J Neurotrauma 1990; 7(2):77–87.
79 Thomas DGT, Lannigan CB, Behan PO. Impaired cellmediated immunity in human brain tumours. Lancet 1975; i:1389–90.
80 Antel JP, Nicholas MK, Bania MB et al. Comparison of T8+ cell-mediated suppressor and cytotoxic functions in multiple sclerosis. J Neuroimmunol 1986; 12:215–24.
81 Rohowsky-Kochan C, Eiman D, Troiano R et al. Decreased suppressor-inducer T lymphocytes in multiple sclerosis and other neurological diseases. J Neuroimmunol 1990; 28:161–6.
82 Freedman MS, Muth KL, Trotter JL et al. Prospective serial analysis of interleukin-2 receptor in relapsing-remitting multiple sclerosis. Neurology 1992; 42:1596–601.
83 Quinn S. Human Trials. Scientists, Investors and Patients In the Quest for a Cure. Cambridge, MA: Perseus Publishing; 2001.
84 Simpson JA. Myasthenia gravis. A new hypothesis. Scot Med J 1960; 5:419–36.
85 Behan PO, Haniffah BAG. The genetics of myasthenia gravis. In: Ed. Lisak RP. Handbook of Myasthenia Gravis and Myasthenic Syndromes. New York: Marcel Dekker Inc (Pub); 1994; 165–91.
86 Shakir RA, Hussien JM, Trontelj JJ. Myasthenia gravis and neuromyelitis optica. J Neuroimmunol 1983; 4:161–5.
87 Aita FJ, Snyder HD, Reichl W. Myasthenia gravis and multiple sclerosis: an unusual combination of diseases. Neurology 1974; 24:72–5.
88 Warren SA, Warren KG. Multiple sclerosis and diabetes mellitus: further evidence of a relationship. Can J Neurol Sci 1982; 9:415–19.
89 Alter M, Sawyer GT, Latham K. The frequency of diabetes mellitus in families of patients with multiple sclerosis. Neurology 1970; 20:619–21.
90 Broadley SA, Deans J, Sawcer SJ et al. Autoimmune disease in first-degree relatives of patients with multiple sclerosis. A UK survey. Brain 2000; 123:1102–11.
91 Heinzlef O, Alamowitch S, Sazdovitch S et al. Autoimmune disease in families of French patients with multiple sclerosis. Acta Neurol Scand 1999; 100:1–5.
92 McDonald WI, Compston A, Edan G et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the International Panel on the diagnosis of multiple sclerosis. Ann Neurol 2001; 50:121–7.
93 Amato MP, Ponziani G, Bartolozzi ML et al. A prospective study on the natural history of multiple sclerosis: clues to the conduct and interpretation of clinical trials. J Neurol Sci 1999; 168:96–106.
94 O’Riordan JI, Thompson AJ, Kingsley DP et al. The prognostic value of brain MRI in clinically isolated syndromes of the CNS: a 10-year follow-up. Brain 1998; 121:495–503.
95 The Multiple Sclerosis Study Group. Efficacy and toxicity of cyclosporine in chronic progressive multiple sclerosis: a randomised, double-blinded, placebo-controlled clinical tria. Ann Neurol 1990; 27:591–605.
96 Likosky WH, Fireman B, Elmore R et al. Intense immunosuppression in chronic progressive multiple sclerosis: the Kaiser study. J Neurol Neurosurg Psychiat 1991; 54:1055–60.
97 The Canadian Cooperative Multiple Sclerosis Study Group. The Canadian cooperative trial of cyclophosphamide and plasma exchange in progressive multiple sclerosis. Lancet 1991; 337:441–6. 98 Hauser SL, Dawson DM, Lehrich JR et al. Intensive immunosuppression in progressive multiple sclerosis: a randomised, three-arm study of high-dose intravenous cyclophosphamide, plasma exchange, and ACTH. N Eng J Med 1993; 308:173–80. 99 Yudkin PL, Ellison GW, Ghezzi A et al. Overview of azathioprine treatment in multiple sclerosis. Lancet 1991; 338:1051–5.
100 Compston A. Treatment and management of multiple sclerosis. In: McAlpine’s Multiple Sclerosis. 3rd edition. London: Churchill Livingston; 1998; 437–98.
101 Dhib-Jalbut S. Mechanisms of action of interferons and glatiramer acetate in multiple sclerosis. Neurology 2002; 58(Suppl 4):S3–9.
102 Calabresi PA. Considerations in the treatment of relapsing-remitting multiple sclerosis. Neurology 2002; 58(Suppl 4):S10–22.
103 Beecher HK. The powerful placebo. JAMA 1955; 159:1602– 6.
104 Behan PO, Melville ID, Durward WF et al. Transfer-factor therapy in multiple sclerosis. Lancet 1976; i:988–90.
105 Behan PO. Dorsal column stimulation in multiple sclerosis. BMJ 1980; 2:1287–8.
106 Mayor S. Researchers claim clinical trials are reported with misleading statistics. BMJ 2002; 324:1353.
107 Clarke M, Alderson P, Chalmers I. Discussion sections in reports of controlled trials published in general medical journals. JAMA 2002; 287:2799–801.
108 Umehara F, Qin YF, Goto M et al. Experimental autoimmune encephalomyelitis in the maturing central nervous system. Transfer of myelin basic protein-specific T line lymphocytes to neonatal Lewis rats. Lab Invest 1990; 62(2):147–55.
109 Oppenheimer DR. Acute encephalopathy in children. In: Eds. Whitty CWM, Hughes JT, MacCallum FO. Virus diseases and the Nervous System. Oxford and Edinburgh: Blackwell Scientific Publications; 1969.
110 Guilhoto LMRR, Osorio CAM, Machado LB. Paediatric multiple sclerosis report of 14 cases. Brain Devel 1995; 17:9–12.
111 Shaw CM, Alvord EC. Multiple sclerosis beginning in infancy. J Child Neurol 1987; 2:252–6.
112 Brandt S, Gyldensted C, Offner H et al. Multiple sclerosis with onset in a two-year-old boy. Neuropaediatrics 1981; 12(1):75–82.
113 Taylor D, Cuendet F. Optic neuritis in childhood. In: Eds. Hess RF, Plant GT. Optic Neuritis Cambridge: Cambridge University Press; 73–85.
114 Berger JR, Sheremata WA, Resnick L et al. Multiple sclerosis-like illness occurring with human immunodeficiency virus infection. Neurology 1989; 39:324–9.
115 Peterson K, Rosenblum MK, Powers JM et al. Effect of brain irradiation on demyelinating lesions. Neurology 1993; 43:2105–12.
116 Oldendorf WH, Cornford EM. A comparison of total body and local spinal cord irradiation in experimental allergic encephalomyelitis. J Neuropathol Exp Neurol 1997; 36:50–61.
117 Levine S, Hoenig EM. Induced localisation of allergic adrenalitis and encephalomyelitis at sites of thermal injury. J Immunol 1968;100(6):1310–18.
118 Aarli JJ, Mork SJ, Myrseth E et al. Glioblastoma associated with multiple sclerosis: Coincidence of induction? Eur Neurol 1989; 29:312–16.
119 Tourtellotte WW, Potvin AR, Baumhefner RW et al. Multiple sclerosis de novo CNS IgG synthesis: effect of CNS irradiation. Arch Neurol 1980; 37:620–4.
120 Cook SD, Devereux C, Troiano R et al. Total lymphoid irradiation in multiple sclerosis: blood lymphocytes and clinical course. Ann Neurol 1987; 22:634–8.
121 Cook SD, Devereux C, Troiano R et al. Effect of total lymphoid irradiation in chronic progressive multiple sclerosis. Lancet 1986; 1:1405–9.
122 Devereux CK, Vidaver R, Hafstein MP et al. Total lymphoid irradiation for multiple sclerosis. Int J Radiat Oncol Biol Phys 1988; 14:197–203.
123 Cook SD, Troiano R, Zito G et al. Deaths after total lymphoid irradiation for multiple sclerosis. Lancet 1989; 2:277–8.
124 Scherer JJ. La ‘Glioblastomatose en plaques.’ J Belge Neurol Psychiat 1938; 38:1–17.
125 Munch-Peterson CJ. Case of disseminated sclerosis and glioma of the brain in the same patient. Acta Psychiat Neurol Scand 1949; 24:599–605.
126 Zimmerman HM, Netsky MG. The pathology of multiple sclerosis. Res Publ Assoc Nerv Ment Dis 1950; 28:271–312.
127 Matthews WB. Epilepsy and disseminated sclerosis. Quart J Med 1962; 31:141–55.
128 Brihaye J, Perier O, Stenuit J. Multiple sclerosis associated with a cerebral glioma. J Neuropath Exp Neurol 1963; 22:128–37.
129 Boyazis RM, Martin L, Bouteille M et al. Images histochimiques et ultrastructurales dans un cas de sclerose en plaques associee a un spongioblastome. Riv Pat Nerv Ment 1968; 88:1–20.
130 Barnard RO, Jellinek EH. Multiple sclerosis with amyotrophy complicated by oligodendroglioma. J Neurol Sci 1967; 5:441–55.
131 Aubert L, Arroyo H, Dumas B et al. Un can d’association entre sclerose en plaques et glioblastome cerebral. Rev Neurol 1968; 119:374–6.
132 Russell DS, Rubenstein LJ. In: Pathology of tumours of the nervous system. 4th edition. London: Edward Arnold (Publishers) Ltd; 1977.
133 Mathews T, Moosy J. Mixed glioma, Multiple sclerosis & Charcot-Marie-Tooth Disease. Arch Neurol 1972; 27:263– 8.
134 Reagan TJ, Frieman IS. Multiple cerebral gliomas in multiple sclerosis. J Neurol Neurosurg Psychiat 1973; 36:523–8.
135 Lynch PG. Multiple sclerosis and malignant gliomas (letter). BMJ 1974; 3:577.
136 Currie S, Urich H. Concurrence of multiple sclerosis and glioma. J Neurol Neurosurg Psychiat 1974; 37:598–605.
137 Kalimo H, Frey H, Raine CS et al. Late-onset malignant astrocytoma in a case of multiple sclerosis. Acta Neuropathol (Berl.) 1979; 46:231–4.
138 Lahl R. Combination of multiple sclerosis and cerebral glioblastoma. Eur Neurol 1980; 19:192–7.
139 Ho KL, Wolfe DE. Concurrence of multiple sclerosis and primary intracranial neoplasms. Cancer 1981; 47:2913– 19.
140 Vieregge P, Nahser HC, Gerhard L et al. Multiple sclerosis and cerebral tumour. Clin Neuropathol 1984; 3(1):10–21.
141 Malmgren RM, Detels R, Verity MA. Co-occurrence of multiple sclerosis and glioma – case report and neuropathologic and epidemiologic review. Clin Neuropathol 1984; 3(1):1–9.
142 Nahser HC, Vieregge P, Nau HE et al. Coincidence of multiple sclerosis and glioma. Surg Neurol 1986; 26:45– 51.
143 Barnard RO, Geddes JF. The incidence of multifocal cerebral gliomas. Cancer 1987; 60:1519–31.
144 Shankar SK, Rao TV, Srivastav VK et al. Balo’s concentric sclerosis: A variant of multiple sclerosis associated with oligodendroglioma. Neurosurgery 1989; 25(6):982–6.
145 Perilongo G, Moras P, Carollo C et al. Spontaneous partial regression of low-grade glioma in children with neurofibromatosis-1: A real possibility. J Child Neurol 1999; 14:352–6.
146 Costa MF, Novis SAP, Filho PN et al. Esclerose Multipla, Ependimoma Medular E Meningioma Intracraniano. Arq Neuropsiquiatr 2000; 58(4):1133–7.
147 Adams RD, Sidman RL. In: Introduction to Neuropathology. New York: McGraw-Hill; 1968; 34.
148 Okun MR, Edelstein LM. Cell reproduction by subdivision? (letter) Lancet 1967; 1:1056–7.
149 Ferner RE, Hughes RAC, Johnson MR. Neurofibromatosis 1 and multiple sclerosis. J Neurol Neurosurg Psychiat 1995; 58:582–5.
150 Masson C, Colombani JM. Neurofibromatosis 1 and multiple sclerosis. Apropos of a case. Rev Neurologique 1997; 153(11):684–6.
151 Alfonso S, Roquer J, Pou A. Multiple sclerosis and neurofibromatosis 1. Neurologia 1996; 11(6):233–5.
152 Pollock M, Calder C, Allpress S. Peripheral nerve abnormality in multiple sclerosis. Ann Neurol 1977; 2:41– 8.
153 Hasson J, Terry RD, Zimmerman M. Peripheral neuropathy in multiple sclerosis. Neurology 1958; 8(7):503–10.
154 Almsaddi M, Bertorini TE, Seltzer WK. Demyelinating neuropathy in a patient with multiple sclerosis and genotypical HMSN-1. Neuromusc Dis 1998; 8:87–9.
155 Griffin JW, Sheikh K. Schwann cell-axon interactions in Charcot-Marie-Tooth Disease. Ann NY Acad Sci 1999; 883:77–90.
156 Eisen A, Paty D, Hoirch M. Altered supernormality in multiple sclerosis peripheral nerve. Muscle Nerve 1982; 5:411–14.
157 Argyrakis A. Ultrastructural changes in peripheral nerves in multiple sclerosis and subacute sclerosing panencephalitis. In: Eds. Bauer HJ, Poser S, Ritter G. Progress in Multiple Sclerosis Research. Berlin: Springer-Verlag; 1980; 360–4.
158 Schoene WC, Carpenter S, Behan PO et al. ‘Onion bulb’ formations in the central and peripheral nervous system in association with multiple sclerosis and hypertrophic polyneuropathy. Brain 1977; 100:755–73.
159 Rosenberg NL, Bourdette D. Hypertrophic neuropathy and multiple sclerosis. Neurology 1983; 33:1361–4.
160 Ro YI, Alexander CB, Oh SJ. Multiple sclerosis and hypertrophic demyelinating peripheral neuropathy. Muscle Nerve 1983; 6:312–16.
161 Eisen A, Yufe R, Trop D et al. Reduced neuromuscular transmission safety factor in multiple sclerosis. Neurology (Minneap) 1978; 28:498–602.
162 Weir A, Hansen S, Ballantyne JP. Single fiber electromyographic jitter in multiple sclerosis. J Neurol Neurosurg Psychiat 1979; 42:1146–50.
163 Hopf HC, Eysholdt M. Impaired refractory periods of peripheral sensory nerves in multiple sclerosis. Ann Neurol 1978; 4:499–501.
164 Hopf HC. Electromyographic study on so-called mononeuritis. Arch Neurol 1963; 9:307–12.
165 Feigen I. Mesenchymal tissues of the nervous system, the indigenous origin of brain macrophages in hypoxic states and in multiple sclerosis. J Neuropathol Exp Neurol 1969; 28:6–23.
166 Feigen I, Ogata J. Schwann cells and peripheral myelin within human central nervous tissues: the mesenchymal character of Schwann Cells. J Neuropathol Exp Neurol 1971; 30:603–12.
167 Cannella B, Pitt D, Marchionni M et al. Neuregulin and erb B receptor expression in normal and diseased human white matter. J Neuroimmunol 1999; 100(1–2):233–42.
168 Huson SM, Harper PS, Compston DAS. Von Recklinghausen neurofibromatosis. A clinical and population study in south-east Wales. Brain 1988; 111:1355–81.
169 Ferner RE, Chaudhuri R, Bingham J et al. MRI in neurofibromatosis 1. The nature and evolution of increased intensity T2 weighted lesions and their relationship to intellectual impairment. J Neurol Neurosurg Psychiat 1993; 56:492–5.
170 Ratner N, Daston MM. Genetic and cellular mechanisms of Schwann cell tumour formation: neurofibromatosis type 1 and neurofibromatosis type 2. In: Eds. Jessen KR, Richardson WD. Glial cell development. 2nd edn. Oxford: Oxford University Press; 2001; 355–8.
171 Waxman SG. Multiple sclerosis as a neuronal disease. Arch Neurol 2000; 57:2–24.
172 Matthew PM, De Stefano N, Narayanan S et al. Putting magnetic resonance spectroscopy studies in context: axonal damage and disability in multiple sclerosis. Seminars in Neurology 1998; 18:327–36.
173 Lucchinetti CF, Brueck W, Rodriguez M et al. Multiple sclerosis: lessons from neuropathology. Seminars in Neurology 1998; 18:337–45.
174 Narayana PA, Doyle TJ, Lai D et al. Serial proton magnetic resonance spectroscopy imaging, contrast enhanced magnetic resonance imaging and quantitative lesion volumetry in multiple sclerosis. Ann Neurol 1998; 43:56– 71.
175 Gonen O, Catalaa I, Babb JS et al. Total brain Nacetylaspartate: a new measure of disease load in multiple sclerosis. Neurology 2000; 54:15–19.
176 Fu L, Matthews PM, De Stefano N et al. Imaging axonal damage in the normal appearing white matter in multiple sclerosis. Brain 1998; 121:103–13.
177 Evangelou N, Esiri MM, Smith S et al. Quantitative pathological evidence for axonal loss in normal appearing white matter in multiple sclerosis. Ann Neurol 2000; 47:391–5.
178 Davie CA, Barker GJ, Webb S et al. Persistent functional deficit in multiple sclerosis and autosomal dominant cerebellar ataxia is associated with axon loss. Brain 1995; 118:1583–92.
179 Arnold DL, Reiss GT, Matthews PM et al. Use of proton magnetic resonance spectroscopy for monitoring disease progression in multiple sclerosis. Ann Neurol 1994; 36:76– 82.
180 Bjartmar C, Kinkel RP, Kidd G et al. Axonal loss in normalappearing white matter in a patient with acute MS. Neurology 2001; 57:1248–52.
181 van Waesberghe JHTM, Kamphorst W, De Groot CJA et al. Axonal loss in multiple sclerosis lesions: magnetic resonance imaging insights into substrates of disability. Ann Neurol 1999; 46:747–54.
182 van Waderveen MAA, Truyen L, van Oosten BW et al. Development of hypointense lesions in T1-weighted spinecho magnetic resonance images in multiple sclerosis: relation to inflammatory activity. Arch Neurol 1999; 56:345– 51.
183 Beaulieu C, Allen PS. Determinants of anisotropic water diffusion in nerves. Magn Reson Med 1994; 31:394–400.
184 Pierpaoli C, Jezzard P, Basser PJ et al. Diffusion tensor MR imaging of the human brain. Radiology 1996; 201:637–48.
185 Hajnal JV, Doran M, Hall As et al. Clinical use of diffusiontensor imaging for diseases causing neuronal and axonal damage. AJNR Am J Neuroradiol 1999; 20:1044–8.
186 Werring DJ, Clar CA, Barker GJ et al. Diffusion tensor imaging of lesions and normal-appearing white matter in multiple sclerosis. Neurology 1999; 52:1626–32.
187 Tivesky AL, Ptak T, Farkas J. Investigation of apparent diffusion coefficient and diffusion tensor anisotropy in acute and chronic multiple sclerosis lesions. AJNR Am J Neuroradiol 1999; 20:1491–9.
188 Sorensen AG, Wu O, Copen WA et al. Human acute cerebral ischaemia: detection of changes by water diffusion anisotropy by using MR imaging. Radiology 1999; 212:785–92.
189 Guo AC, Petrella JR, Kurtzberg J et al. Evaluation of white matter anisotropy in Krabbe disease using diffusion tensor MR imaging: initial experience. Radiology 2001; 218:809– 15.
190 Fillipi M, Rocca MA, Minicucci L et al. Magnetization transfer imaging of patients with definite MS and negative conventional MRI. Neurology 1999; 52:845–8.
191 Iannucci G, Tortorella C, Rovaris M et al. Prognostic value of MR and magnetization transfer imaging findings in patients with clinically isolated syndromes suggestive of multiple sclerosis at presentation. AJNR Am J Neuroradiol 2000; 21:1034–8.
192 Guo AC, MacFall JR, Provenzale JM. Multiple sclerosis: diffusion tensor MR imaging for evaluation of normal appearing white matter. Radiology 2002; 222:729–36.
193 Inglese M, Salvi F, Iannucci G et al. Magnetization transfer and diffusion tensor MR imaging of acute disseminated encephalomyelitis. AJNR Am J Neuroradiol 2002; 23:267– 72
194 Tortorella C, Viti B, Bozzali M et al. A manetization transfer histogram study of normal appearing brain tissue in MS. Neurology 2000; 54:186–93.
195 Rovaris M, Viti B, Ciboddo G et al. Brain involvement in systemic immune mediated diseases: magnetic resonance and magnetisation transfer imaging study. J Neurol Neurosurg Psychiatry 2000; 68:170–7.
196 van Waesberghe JHTM, van Walderveen MAA, Catelijns JA et al. Patterns of lesion development in multiple sclerosis: longitudinal observations with T1-weighted spin-echo and magnetization transfer MR. AJNR Am J Neuroradiol 1998; 19:675–83.
197 Huppi PS, Murphy B, Maier SE et al. Microstructural brain development after perinatal cerebral white matter injury assessed by diffusion tensor magnetic resonance imaging. Pediatrics 2001; 107:455–60.
198 Majoie CB, Akkerman EM, Blank C et al. Mitochondrial encephalomyopathy: comparison of conventional MR imaging with diffusion-weighted and diffusion tensor imaging: case report. AJNR Am J Neuroradiol 2002; 23:813–16.
199 Fillipi M, Cercignani M, Inglese M et al. Diffusion tensor magnetic resonance imaging in multiple sclerosis. Neurology 2001; 56:304–11.
200 Confraveux C. Relapses, progression, inflammation and neurodegeneration in multiple sclerosis: a changing view. Advances in Clinical Nerosciences and Rehabilitation 2002; 2(1):7–9.
201 Betemps EJ, Buncher CR. Birthplace as a risk factor in motor neurone disease and Parkinson’s disease. Int J Epidemiol 1993; 22(5):898–904.
202 Poser CM. Trauma to the central nervous system may result in formation or enlargement of multiple sclerosis plaques. Arch Neurol 2000; 57:1074–6.
203 Cardoso F, Jankovic J. Peripherally induced tremor and parkinsonism. Arch Neurol 1995; 52:263–70.
204 Jankovic J. Post-traumatic movement disorders: central and peripheral mechanisms. Neurology 1994; 44:2006– 14.
205 Sibley WA, Bamford CR, Clark K et al. A prospective study of physical trauma and multiple sclerosis. J Neurology Neurosurgery Psychiatry 1991; 54:584–9.
206 Kurtzke JF, Beebe GW. Epidemiology of amyotrophic lateral sclerosis: 1. A case-control comparison based on ALS deaths. Neurology 1980; 30:453–62.
207 Kelly PJ, Shapiro BE. Amyotrophic lateral sclerosis and other motor neurone disorders. In: Eds. Bachelor T, Gudkowicz MR. Principles of Neuroepidemiology. Boston: Butterworth; 2001; 265–87.
208 Morris HW, Moriabadi NF, Lees AJ et al. Parkinsonism following electrical injury to the hand. Movement Disorders 1998; 13(3):600–02.
209 Chataway J, Mander A, Robertson N et al. Multiple sclerosis in sibling pairs: an analysis of 250 families. J Neurology Neurosurgery Psychiatry 2001; 71:757–810.
210 Wolfgram F. What if multiple sclerosis isn’t an immunological or a viral disease? The case for a circulating toxin. Neurochem Res 1979; 4:1–14.
211 van Noort J, van Sechel AC, Bajramovic JJ et al. The small heat-shock protein and ß crystalline as candidate autoantigen in multiple sclerosis. Nature 1995; 375:798– 810.
212 McGrath J. Does ‘imprinting’ with low prenatal vitamin D contribute to the risk of various adult disorders? Med Hypotheses 2001; 56(3):367–71.
213 Palo J, Duchesne J, Wikstrom J. Malignant disease among patients with multiple sclerosis. J Neurol 1977; 216:217– 22.
214 Spaar FW, Wikstrom J. Multiple sclerosis and malignant neoplasm of the central nervous system. A clinical anatomical report of three cases. J Neurol 1978; 218:23– 33.
215 Scully RE, Galdabani JJ, McNeely BU. Weekly clinicopathological exercise-Case 44. N Engl J Med 1978; 299:1060–7.
216 De Caso R. Esclerose multipla, ependimoma medular e meningioma intracraniano. Arq Neuropsiquiatr 2000; 58:1133–7. 265