SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire area. When sera of siblings were examined at the same time, the titres of measles antibody for all three antigens fell between the means of the patients and controls; and, in Denmark, OfTner et al. (1973) had similar findings when they used a gel precipitation method of measuring measles antibody. This observation may reflect shared determinants at the major histocompatibility complex, e.g. Ir genes, between patients and siblings. As MS is also associated with determinants of this gene complex (Arnason et al. 1974; Jersild et al. 1975), it is possible that susceptibility to MS and immune responsiveness to measles virus are different manifestations of a certain genetic constitution. While attention has been focused on measles virus, surveys have been made in many centres of antibodies to a variety of viruses including mumps, the influenza viruses, the parainfluenza viruses, rubella, adenovirus, respiratory syncytial virus, the three poliovirus types, several group A and group B Coxsackie viruses, "BK" virus (a human papova virus), Epstein-Barr (EB) virus and canine distemper virus (e.g. Brody et al. 1972; Salmi, 1973). Raised antibody titres have been found to herpes simplex and varicella zoster (Ross et al. 1965; Brody et al. 1971) and in the case of the latter the increase has been significant. In Belfast, using the immunofluorescent technique, we have made serological surveys of virus-specific IgG and IgM in MS patients. Presence of the latter antibody may be presumptive evidence of the persistence of virus antigen in the patient (Haire & Hadden, 1970; Connolly et al. 1971). Levels of measles and herpes simplex IgG were significantly increased in 57 MS patients compared with 57 age- and sex-matched healthy controls, while there was no difference between the groups of IgG for mumps, rubella and varicella zoster viruses (Haire et al. 1973a). Sera of 9/56 of these patients contained IgM which gave weak immunofluorescent staining of the membrane of fixed roeasles-virus-infected tissue-culture cells, but there was no IgM to the viruses referred to above. Seventeen sera gave an IgM "fibrillar" staining pattern of uninfected tissueculture cells (Plate HI: A) similar to that associated with acute or persisting virus infection (Haire, 1972; Samuel et al. 1976). Subsequently, using fresh unfixed virus-infected tissue-culture cells as antigen, we detected measles IgM of higher titre in 11/30 different MS patients (Plate DI: B) (Haire et al. 1973b), and during the past two years this antibody has been detected in 93/203 additional patients' sera (M Haire and A M McKee, unpublished data). We have also found that in individual patients there is a fluctuation in appearance and titre of measles IgM, which, therefore, may occur in an even higher proportion of patients. Measles IgM was shown in IgM fractions obtained by gel filtration of sera of six MS IgM-positive patients in the category described as "chronic probable" (category 2) (Millar, 1971; Harmon et al. 1975) and in similar fractions of sera of patients with acute and early signs of MS, who had presented initially with optic neuritis (A M McKee, W M Hutchinson and M Haire, unpublished data). By treatment of sera with 2-mercaptoethanol, Karaseva et al. (1974) showed, by haemagglutination inhibition, measles 19 S antibodies in 30/105 MS patients and none in a large control group, and, as we have found recently also, the titre was not constant in each patient. The method was checked by finding measles-specific antibody in macroglobulins separated by gel filtration with Sephadex 200. This is the first confirmation we have seen of our findings of measles IgM in a proportion
SIGNIFICANCE OF VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS MARGARET HAIRE MD MRCPath Department of Microbiology and Immunobiohgy The Queen's University of Belfast 1 Serological surveys of virus-specific antibodies in multiple sclerosis 2 Virus-specific antibodies in cerebrospinalfluidof multiple sclerosis patients 3 Relation between cerebrospinal fluid antibodies and immunoglobulins 4 Significance of measles virus antibody findings 5 Summary References The failure to isolate virus from multiple sclerosis (MS) patients by cultural methods or by animal inoculation, or to identify by electron microscopy particles specific for the disease (Johnson, 1975), has increased the importance of the study of virus antibodies, in the hope that the immunological characteristics might point towards the causative agent. Since surveys of virus antibodies in serum have recently been fully discussed (Fraser, 1975), emphasis in this review will be given to reports that have led to the intensive investigation of measles antibodies in both serum and cerebrospinal fluid (CSF). Further investigations, in relation to measles and other antibodies, which require to be made, will be discussed. 1
Serological Surreys of Virus-Specific Antibodies in Multiple Sclerosis
Much effort has been expended on surveys of virus-specific antibodies in the sera of patients with MS and in those of controls to determine whether different responses in the groups might indicate that a virus or several viruses are involved in the pathogenesis of the disease. This has stemmed from the initial finding by Adams & Imagawa in 1962 that measles antibody titres were increased in MS patients compared with controls; subsequent reports, reviewed by Brody et al. (1972) and more recently by Fraser (1975), have supported this observation. In a discussion of 27 reported studies, using different laboratory techniques, Fraser (1975) noted significantly higher levels of measles antibody of the order of a 2-2$-fold excess in MS patients over controls. From our own studies (Haire et al. 1973a, b) and thereportsof many others, the scatter of individual titres has been shown to be small, and the antibody titre in each patient has not been shown to be of any diagnostic significance or to bear anyrelation to the clinical activity ofthe disease. In Sweden and Finland, workers have made an important contribution by their use of different measles antigens, and they have found significantly higher antibody titres to the three separate structural components, nucleocapsid, haemagglutinin and haemolysin (Salmi et al. 1972a; Salmi et al. 1973) in MS patients compared with healthy persons resident in the same 40 Downloaded from https://academic.oup.com/bmb/article-abstract/33/1/40/299852 by Insead user on 14 August 2018
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SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire used in the survey and MS, a conclusion already made by Nikoskelainen et al. (1972). Autoantibodies are commonly associated with virus infections (Holborow, 1972), but we have been unable to correlate them with virus antibodies. We have shown an inCanine distemper creased incidence and higher titres of virus smooth-muscle antibody (SMA),"fibrillar'* antibody to HEp 2 tissue-culture cells and 20.1 to cultured rat cerebellar cells, in the 11.9 IgM class, in 11 MS sera lacking measles 15.2 IgM, compared with age- and sex-matched normal control sera, while there was no difference in the IgG class. Antibodies detected by the three antigens did not correspond, though absorption with both human uterus and normal brain powderreducedthe titres considerably (PlateTV: C). The specificities are unknown, but the SMA IgM autoantibody may be evidence of active cell membrane changes in this group of patients, all clinically free of secondary complications such as infection, and is similar to that described in infectious and malignant diseases by Holborow (1972). Further work is necessary to test a larger series of MS sera including those with measles IgM, searching especially for antibodies to defined central nervous system (CNS) antigens. The only report of IgM antibody activity in MS sera, of which 1 am aware, is that of Dowling et al. (1968) who have shown 19 S and 7 S demyelinating activity in MS sera on cultured neonatal mouse cerebellum. Areviewof antibodies in MS sera would be incomplete without reference to neutralizing antibodies to the MS-associated agent (MSAA) (Henle et al. 1975). Carp et al. (1972) first described the suppression of polymorphonuclear neutrophils (PMN) in mice following inoculation of material from MS patients, and Henle et al. (1975) have confirmed that there is a transmissible MSAA and have extended the work to include serological evidence. The antibodies, of IgG class, are found in patients' sera, in sera of theirrelativesand of nursing personnel and in a considerable proportion of sera from East Africa, where MS is unknown. Sera from healthy American blood donors and from patients with virus infections seldom have such antibodies.
TABLE I. Geometric mean titres of antibody* to measles, rinderpest and canine distemper viruses in eight multiple sclerosis (MS) sera and five acute measles sera Number and type of sera
Immunoglobulin
Antibody to Measles virus
Rinderpest virus
MS (8)
IgG IgM
56.9 26.1
23.9 4.2
Acute measles (5)
IgM
34.8
10.0
* Titres of antibody are expressed as reciprocals
of MS sera at a rate similar to that which we reported in 1973 (Haire et al. 1973a,b). The antigen responsible for measles IgM activity in MS differs from that found in acute measles infection (Fraser et al. 1974) and its specificity is being investigated. In one investigation in collaboration with F Brown (Haire et al. 1974a), eight MS sera andfivesera obtained early in uncomplicated measles infection (all possessing IgM activity to the membranes of measles-virus-infected cells) were tested for both IgM and IgG antibodies, using cells infected with the cross-reacting viruses, rinderpest and canine distemper (Imagawa, 1968). With both groups of sera the titres against measles were highest (Table I), which suggests that in MS patients the antigenic stimulus is measles virus or one of its components rather than the other two related viruses. Our own and other reported findings of raised titres of antibody to herpesviruses in MS patients prompted us to make a serological survey of IgG and IgM antibodies to cytomegalovirus, varicella zoster, herpes simplex and EB viruses. No significant difference was found in the mean titres of the IgG antibodies between the MS patients and the two control groups (Table II). While neither EB IgM nor cytomegalovirus IgM was found, several sera had low titre IgM to one or both of the other viruses with equal distribution in the three study groups. As both herpes simplex and varicella zoster are latent viruses for man, we did not attach any importance to thisfinding,which confirmed an earlier observation regarding herpes simplex IgM (Millar et al. 1971). From our results we decided that there was no obvious connexion between the herpesviruses
2 Virus-Specific Antibodies in Cerebrospinal Fluid of Multiple Sclerosis Patients Studies of virus antibodies in CSF of MS patients have been restricted by limited quantities obtainable from patients, by the lack of suitable control specimens and by insensitive techniques not capable of detecting the small amount of antibody which might be present. Despite these limitations certain important studies have been reported during the pastfiveyears and are summarized in Table m . Brown et al. (1971) in their series also tested 65 MS CSF and 35 of the control CSF for antibodies to other myxoviruses including influenza A and B, parainfluenza 1-4, respiratory syncytial, rubella and mumps viruses, and while a few of thefluidshad a low titre of antibody, the outstanding finding was the high incidence and significantly highertitresof measles antibody detected by the sensitive mixed haemadsorbing antibody test. In 33 MS and 17 control patients, for whom matching CSF and serum specimens were available, statistical analysis did not show a significant over-all correlation between
TABLE II. Geometric mean titres of virus-specific IgG antibody* in patients with MS, normal control subjects and neurological control patients Virus
Herpes simplex Varicella zoster Cytomegalovl rus EB virus Number
Group MS patlentsf
Normal subjects!
Neurological controls^
45.8 21.9 9.3 31.4
31.9 11.9 11.3 24.1
53.9 15.5 18.5 33.3
39
39
35
* Titres of antibody are expressed as reciprocals t Age- and sex-matched subjects (16 males, 23 females) X Slightly older controls (16 males, 19 females)
41 Vol. 33 No. 1
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SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire CSF and serum antibody levels. Salmi et al. (1972b) showed that the one gel precipitation line produced by CSF from 2/15 MS patients against measles antigen had a reaction of identity with one of several lines produced by the CSF of a patient with subacute sclerosing panencephalitis (SSPE), in which condition measles antigen has been found in brain and extremely high titres of measles antibody have been measured in CSF
(Connolly et al. 1968). Complement-fixing (CF) antibody to 27 other viruses tested was not found in these two CSF. During 1973 it appeared possible that vaccinia virus, used for immunization against smallpox, might be involved in some way in the aetiology of MS, after Kempe et al. (1973) found neutralizing antibody to vaccinia in 30% of MS CSF, with less than 3 % in CSF of the neurological control group and none in a normal control group (medical students). In order to confirm this finding, Brown et al. (1973) tested CSF from 127 TABLE III. Studies of virus antibodies in cerebrospinal fluid (CSF) MS patients in France and from 78 of MS patients patients with other neurological disorders, and detected vaccinia antibody in none of Virus Antibody Test positive in Comment the MS patients and in one patient in the control group. Exchange of specimens beMS Neurological Normal controls controls tween the two laboratories ensured that there were no technical differences. One Mixed Measles* 94/119(79%) 51/112(45%) Increased frequency In possible explanation for the finding was MS; significantly higher haemadsorbing titres in MS that in France, where vaccination is per_ Measles* Gel predpitating 2/15(13%) Gel precipitation lines formed at 12 years of age without subwere identical with 1/3 sequent boosters, the chance of detectable lines produced by subacute scteroslng antibody in serum at the time of obtaining panencephalitis CSF CSF from MS patients would be minimal. Vacdnia Neutralizing 56/187(30%) 3/87 (3%) USA population which 0/30 had received fairly Kempe's specimens were from patients in extenslvo vaccination* the USA where, on the other hand, until Vacdnia Neutralizing 0/127 1/78(1%) French population which received vaccination at very recently, fairly extensive and frequent 12 years; boosters vaccination was carried out; so it is reasonrare* Paralnfluenxa Neutralizing 0/62 0/36 able to assume that serum antibody levels Negative findings type 1* would be high and that the CSF antibodies Measles* /•I6/25 (64%) 0/17 \ Measles, herpes simplex may be due to leakage across the bloodHerpes simplex* I 9/25(36%) 0/171 FAt IgG and mumps IgM was Mumps* i 0/25 0/17/ brain barrier. Brown et al. (1973) also tested not found in CSF Rubella* I 0/11 0/I6J 62 MS and 36 control CSF of this series for 19/301 (63%) I/30II.111 Measles 1/30" (3%) [. 9/19—measles IgM in serum neutralizing antibody to parainfluenza type li. Patient with jreneral 1 virus, with completely negative results. paralysis of the in«n«—measles IgM in serum In Belfast we have not found IgM antilil. Patient with brain body in CSF, of either MS patients or tumour and FAt IgG morbilliform rash control subjects, to any viruses we have iv. "Normal" control found to have tested, viz. measles, mumps, herpes simplex spastic paraparesls and rubella. This is not surprising, as Link •f extensor plantar & Muller (1971) did not show an increase responses Herpes simplex " 11/30(37%) 7/30 (23%) 2/30(7%) I 6/18(33%) Rubella' j 2/21 (9%) in total IgM in 64 MS CSF, though their 1/16(6%) Measles* 13/30 (43%) 2/30(7%) Haemagglutinin 2/30(7%) -v technique did reveal an increase in 9% of inhibiting (CSF and serum] CSF of 39 patients with infections of the Nudeocapsid Reduced serum: CSF 10/30 (33%) 0/30 0/30 complement ratios in 12/30 MS CNS. By indirect immunofluorescence we fixing patients; none In either HaemoTysln have found IgG antibody specific for 14/19(74%) 2/18(11%) 3/19 (16%) J control group inhibiting measles, herpes simplex and rubella viruses Measles* 23/80(29%) 1/97(1%) Haemagglutinin (Haire et al. 1973a,b) in the CSF of MS Inhibiting 0/98 Ribonudeoprotein 16/80(20%) Measles and rubella antipatients, but that to measles predominated gel predpitating bodies In positive cor1 relation with IgG and Haemolysin and in several separate studies the preval41/79(52%) 6/92 (6%) Inhibiting - ( higher ratio of ence has been consistent (Haire et al. 1974b). Haemaggtutlnin Rubella* 2/94(2%) lgG:albumin 18/84(21%) inhibiting We have tested CSF and serum obtained at Measles — ( 57%S the same time from 30 MS patients, 30 l9 Rubella Local production in CNS patients with other neurological diseases 1 7i Mumps Reduced _ to any of J '^%¥ and 30 " normal" controls who were patients - 1 oftheantibodies Herpes simplex serum:CSF ratiot i 1 l%j — viruses in 7 1 % MS - r patients type \J undergoing myelography for suspected proParainfluenza — 1 3%S type 1^ lapsed intervertebral disk, but we did not establish a positive correlation between the * Laboratory exchange of sped m e n checked that the results of Kempe et of. (1973) and of Brown et of. (1973) presence of measles IgG in CSF and were correct measles IgM in serum in MS (Haire et al. PA: Indirect Immunofiuorescence for IgG antibody Several different serological tests were employed 1973b), though it is interesting that 9/11 150 patients were studied patients whose sera had measles IgM had References: also measles IgG in CSF. In this series, o: Brown et of. 1971 d: Brown et of. 1973 g: Norrby et al. ]974a b: Salmi et of. 1972b e: Haire et al. 1973a Salmi et at. 1974 CSF from 10/30 patients had IgG to both c: Kemp* et of. 1973 f: Haire et al. 1973b / : Norrby et of. 1974b 0
4
/
7
7
7
7
- 1 -I
i
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Br. Med. Bull. 1977
PLATE III
SIGNIFICANCE OF VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS Margaret Haire (FIG. A-C)
A. "Fibrillar" staining pattern seen when fixed tissue-culture cells are treated with MS serum and anti-human IgM conjugated with fluorescein isothiocyanate (FITC) (mif nificition x 550)
B. Membrane staining of measles-vlrus-infected tissue-culture cells, seen when live infected cells are treated with MS serum and antihuman IgM conjugated with FITC (mainificacion x 550)
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PLATE IV
SIGNIFICANCE OF VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS (continued)
CON
CON
MS
MS
CON
ill dii)
di)
MS
CON
MS
CON
-1 (ii)
(i)
c
MS •
CON
(iii) CON
MS
CON
MS
log,
o
>
-2-_
S
:
ant
-9 o
IILI.
• 1
Ill
H
1m (i)
ill (ii)
• •
(iii)
C. Titres of IgM antibody in 11 MS sera and In sera of age- and sex-matched controls ( C O N ) before absorption (I), after absorption with human uterus (II), and after absorption with normal brain powder (iii). Antigens used are: ( a ) composite blocks of rat liver, stomach and kidney to detect smooth-muscle antibody; ( b ) HEp 2 tissue-culture cells; and (c) cultured cells from cerebellum of newborn rats. Cultured cells are acetone fixed
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SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire neutralizing activity against the MSAA, whilefluidsfrom two control patients had no such effect. Two of three sera tested from these four MS patients also contained neutralizing activity. Information regarding autoantibodies in CSF is scanty. While Laurell & Link (1972) showed CF antibodies reacting with crude MS brain in 6/12 CSF from MS patients and in 0/17 CSF from neurological control patients, they also showed CF antibodies detectable at a lower titre in 5/6 CSF when normal brain antigen was used. As in the case of sera, further work is needed to detect antibodies to defined CNS antigens in CSF of MS patients, and to relate detectable antibody to the several membrane viruses and the MSAA which have already been shown to possess antibody activity within the CNS.
measles and herpes simplex viruses, and serum: CSF ratios of antibody in these 10 patients indicated lower ratios in the case of measles virus; but we are not prepared to conclude that this means active growth of virus in the CNS (Fraser, 1975). Clarke et al. (1965) found ratios of serum:CSF poliovirus antibody to be lower in MS patients compared with controls; and indeed this reduced ratio tended to be associated with higher globulin concentrations in the CSF, indicating that in MS at least some of they-globulin in CSF is derived from serum antibody. The report by Connolly et al. (1968) of significantly reduced ratios of titres of serum:CSF measles antibody in the three patients with SSPE, in whose brains measles antigen was found by immunofluorescence, prompted Norrby et al. (1974a) to use this method to seek evidence for measles antibody production in the CNS of MS patients. They compared the ratios of serum:CSF antibodies to different measles virus components with the ratios to two reference viruses, poliovirus and adenovirus, both unlikely to be involved in CNS disease. The study groups were carefuily selected; the CSF of 30 MS patients had two or more extra y-globulin bands (oligoclonal IgG) and the serum electrophoretic pattern was normal. Twenty-six of these patients had raised relative IgG concentration in CSF, while the IgG content of the CSF of the 30 neurological control patients was normal, though 26 of these 30 patients showed a slight to moderate increase in CSF total protein, suggesting damage to the bloodrbrain barrier. The "healthy" control group consisted of 30 patients with minor psychiatric disorders, without apparent CSF abnormality. Twelve of the 30 MS patients showed a significantly reduced ratio of serum: CSF antibody to measles haemagglutinin and measles nucleocapsid, with normal ratios in most cases to the two reference antigens. By measurement of antibody to measles haemolysin 5/19 MS patients studied also showed a reduction. None of the 60 control patients had this reduction, and, as the patients with neurological disorders had moderate damage to the blood-brain barrier, Norrby et al. (1974a) decided that the reduced ratios in MS are not due to selective passage of antibodies to the CNS, but rather to active production within the CNS. Most of the studies from the Scandinavian workers have been concerned with CSF antibodies to measles virus, but two later reports focus attention on rubella and other viruses. In Finland, Salmi et al. (1974) found antibodies in CSF to measles haemagglutinin, measles haemolysin, measles ribonucleoprotcin and rubella haemagglutinin in 28%, 51%, 20% and 20% of MS patients, respectively, with very few of the neurological control patients having any detectable antibody. Both measles and rubella antibodies were in positive correlation with higher levels of IgG and elevated IgG: albumin ratios in CSF specimens, suggesting that in MS some of these antibodies are synthesized within the CNS. A report based on a combined study of 150 MS patients from Sweden, Finland and Norway showed reduced ratios of titres of serum:CSF antibody, interpreted as indicating local production in the CNS, of antibody against measles virus in 57 %, against rubella virus in 19 %, against mumps virus in 15 %, against herpes simplex type 1 virus in 11 % and against parainfluenzatypelvirusin3%. Seventy-one per cent of them showed evidence of local production of antibody in the CNS against at least one virus (Norrby et al. 1974b). Henle et al. (1975) have shown that CSF from four patients with MS whose sera induced depression of PMN in experimental animals, implying the presence of the MSAA, had strong
3 Relation between Cerebrospinal Fluid Antibodies and Imnumoglobulins The presence of oligoclonal IgG in CSF and its absence from serum is very important in diagnosis in MS (Link, 1967), and several of the features of oligoclonal IgG may also give clues in the search for a cause of the disease. The location in both CSF and in MS brains (link, 1972) and the constancy of the band pattern during the course of the disease (Olsson & Link, 1973) may indicate that a responsible antigen, present in the CNS, is continually active. All attempts to investigate its specificity have been restricted to the detection of measles antibody in these bands. In one patient, Panelius & Salmi (1973) related both measles antihaemagglutinin (HI) and CF antibody with one of four bands, and HI antibody with another. Neither antibody was found in the other two bands, and tests were not carried out for antibody to different viruses. Vandvik & Degr6 (1975) found that one or two of several (up to eight) bands were associated with measles gel-precipitating antibody, and they speculated that the non-reactive bands may be antibodies directed against other virus components. Norrby & Vandvik (1975) succeeded, in the case of SSPE, by repeated absorptions with highly concentrated and purified measles virus, in removing almost completely all the oligoclonal IgG and in demonstrating that different bands showed antibody to different measles components. However, using a similar type of absorption of MS CSF they found it possible only in exceptional cases to alter the band pattern. Therefore, the presumed antibody activity of the major part of the oligoclonal IgG has still to be accounted for. Determination of the specificity of this antibody is essential in order to find an antigen which is very likely to be closely connected with the immunopathogenesis of the disease. 4 Significance of Measles Virus Antibody Findings The various surveys of serum antibodies show clearly that MS patients have a hyperactivity to measles virus, and reaction with three separate structural components of this virus (Salmi et al. 1972a; Salmi et al. 1973) adds strength to the view that measles virus may in some way be involved in the pathogenesis of the disease. Measles IgM, found by two groups of workers (Haire et al. 1973a,b; Karaseva et al. 1974), points to a continuing antigenic stimulus; and the greater IgM response in patients to measles rather than to the other viruses of the group, canine distemper and rinderpest viruses, further 43
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VoU33 No. 1
SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire implicates measles in some way. Identification of the particular antigen involved is necessary, as it differs from that of a regular measles infection (Fraser et al. 1974), and until measles virus is shown in biopsy material from patients, in circulating leucocytes or in autopsy material, analysis of serum antibodies or search for immune complexes are the only feasible methods of achieving this. One clue may be that the IgM SMA may be stimulated by an immunologically active membrane antigen, resulting from virus-membrane interaction in the patient While antibody to measles virus predominates in CSF in MS patients, Norrby & Vandvik (1975) did not find a clear correlation between bands of oligoclonal IgG and measlesvirus-specific activity in all patients. In order to explain these bands, for whose presence a responsible antigen or antigens have not been found, it would be worth searching for activity in them to rubella, mumps and herpes simplex viruses, all of which have been shown to produce antibody in CSF of MS patients (Haire et al. 1973b; Norrby et al. 1974b); activity to defined CNS antigens should also be investigated. Assessment of antibody to the MSAA, in both serum and CSF, will be of real value only when a simpler and more reproducible method of assay has been found. If a better assay is developed, full evaluation of the role of MSAA in MS will
be necessary. First, this agent may cross-react with measles, causing increased antibody levels in patients. Secondly, it may affect the cellular immune mechanisms, with resulting elevation of humoral antibody to measles, or, thirdly, it may be an independent infectious agent in some patients. 5 SuDsroary Surveys of virus-specific antibodies in sera and CSF of MS patients give evidence that measles virus may be involved in pathogenesis. The specificity of antibody activity of oligoclonal IgG in CSF will give more precise knowledge of antigens active in MS. ACKNOWLEDGEMENTS
I wish to thank D r J H D Millar, Dr Michael Swallow and Dr J A Lyttle, who have made clinical specimens available. The work reported here has been carried out with the assistance of Dr W M Hutchinson, Mrs Alison McKee, Mrs Patricia McCullough, Mrs Ann Simpson, Miss Caroline Simpson and Mr S A McMillan. Mr McMillan kindly supplied thefiguresfor Plates HI, IV. Research work in multiple sclerosis in the Department of Microbiology and Immunobiology, The Queen's University of Belfast, is supported by a Programme Grant from the Medical Research Council.
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Adams J M & Imagawa D T (1962) Proc. Soc. Exp. Biol. Med. I l l , 562-566 Arnason B G W, Fuller T C, Lehrich J R & Wray S H (1974) / . Neurol. Set. 22,419-428 Brody J A, Sever J L & Henson T E (1971) / . Am. Med. Assoc. 216, 1441-1446 Brody J A, Sever J L, Edgar A & McNew J (1972) Neurology, 22, 492-^99 Brown P, Cathala F, Gajdusek D C & Gibbs C J Jr (1971) Proc Soc. Exp. Biol. Med. 137, 956-961 Brown P, Cathala F & Gajdusek D C (1973) Proc. Soc. Exp. Biol. Med. 143, 828-829 Carp R I, Licursi P C, Merz P A & Merz G S (1972) / . Exp. Med. 136, 618-629 Clarke J K, Dane D S & Dick G W A (1965) Brain, 88, 953-962 Connolly J H, Allen I V, Hurwitz L J & Millar J H D (1968) Q.J.Med. 37, 625-644 Connolly J H, Haire M & Hadden D S M (1971) Br. Med. J. 1,23-25 Dowling P C, Kim S U, Murray M R & Cook S D (1968) /. Immunol. 101,1101-1104 Fraser K B (1975) In: Davison A N, Humphrey J H, Iiversedge L A, McDonald W I & Porterfield J S, ed. Multiple sclerosis research, pp. 53-79 (Proceedings of a joint conference held by the Medical Research Council and the Multiple Sclerosis Society of Great Britain and Northern Ireland, 17-18 October 1974). HMSO, London Fraser K B, Shirodaria P V & Haire M (1974) Med. Microbiol. Immunol. 160, 221-230 Haire M (1972) CUn. Exp. Immunol. 12, 335-341 Haire M & Hadden D S M (1970) Br. Med. J. 3, 130-132 Haire M, Fraser K B & Millar J H D (1973a) CUn. Exp. Immunol 14,409-416 Haire M, Fraser K B & Millar J H D (1973b) Br. Med. J. 3, 612-615 Haire M, Underwood B O & Brown F (1974a) Med. Microbiol. Immunol. 160,234 Haire M, Millar J H D & Merrett J D (1974b) Br. Med. J. 4, 192-193 Hannon R, Haire M. Wisdom G B & Neill D W (1975) /. Immunol. Methods, 8, 29-36 Henle G, Koldovsky U, Koldovsky P, Henle W, Ackennann R & Haase G (1975) Meet. Immun. 12,1367-1374 Holborow E J (1972) Proc. R. Soc Med. 65,481-484 Imagawa D T (1968) Prog. Med. Virol. 10,160-193 Jersfld C, Dupont B, Fog T, Platz P J & Svejgaard A (1975) Transplant. Rev. 22,148-163
Johnson R T (1975) In: Davison A N, Humphrey J H, Liversedge L A, McDonald W I & Porterfield J S, ed. Multiple sclerosis research, pp. 155-183 (Proceedings of a joint conference held by the Medical Research Council and the Multiple Sclerosis Society of Great Britain and Northern Ireland, 17-18 October 1974). HMSO, London Karaseva I A, Khozinskil V I, Chumakov M P, Andreeva L S, Petelin L S, Fridman A Ya, Rostovtseva R I, Smirnov Yu K & Graevskaya N A (1974) Tr. Inst. Polh. Virusn. Entsef. 22,78-83 Kempe C H, Takabayashi K, Miyamoto H, Mclntosh K, Tourtellotte W W & Adams J M (1973) Arch. Neurol. (Chicago) 28,278-279 Laurell A B & link H (1972) Acta Neurol. Scand. 48, 461^*66 link H (1967) Acta Neurol. Scand. suppl. no. 28 link H (1972) / . Neurol. Scl. 16,103-114 link H & MflUer R (1971) Arch. Neurol. (Chicago) 25, 326-344 Millar J H D (1971) Multiple sclerosis: a disease acquired in childhood, p. 3. Thomas, Springfield, 111. Millar J H D, Fraser K B, Haire M, Connolly J H, Shirodaria P V & Hadden D S M (1971) Br. Med. J. 2, 378-380 Nikoskelainen J, Panelius M & Salmi A [A] (1972) Br. Med. J. 4,111 [Letter] Norrby E & Vandvik B (1975) Med. Microbiol. Immunol. 162, 63-72 Norrby E, link H & Olsson J E (1974a) Arch. Neurol. (Chicago) 30, 285-292 Norrby E, Link H, Olsson J E, Panelius M, Salmi A[A] & Vandvik B (1974b) Infect. Immun. 10,688-694 Oflher H, Ammitztwll T & Clausen J (1973) Acta Pathol. Microbiol. Scand. 81B, 157-164 Olsson J E & link H (1973) Arch. Neurol. (Chicago) 28, 392-399 Panelius M & Salmi A A (1973) Acta Neurol. Scand. 49, 266-268 Ross C A C, Lenman J A R & Rutter C (1965) Br. Med. J. 1, 226-229 Salmi A A (1973) Am. CUn. Res. 5, 319-329 Salmi A A, Panelius M & Norrby E (1972a) Lancet, 2,1088-1089 [Letter] Salmi A A, Panelius M, Halonen P, Rinne U K & Penttinen K (1972b) Br. Med. J. 1,477-479 Salmi A [A], Gollmar Y, Norrby E & Panelius M (1973) Acta Pathol. Microbiol. Scand. 81B, 627-334 Salmi A [A], Panelius M & Vainionp&a R (1974) Acta Neurol. Scand. 50,183-193 Samuel M, Shirodaria P V & McMillan S A (1976) CUn. Exp. Dermatol. 1,269-274 Vandvik B & Degre M (1975) /. Neurol. Sd. 24, 201-219
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SIGNIFICANCE OF VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS MARGARET HAIRE MD MRCPath Department of Microbiology and Immunobiohgy The Queen's University of Belfast 1 Serological surveys of virus-specific antibodies in multiple sclerosis 2 Virus-specific antibodies in cerebrospinalfluidof multiple sclerosis patients 3 Relation between cerebrospinal fluid antibodies and immunoglobulins 4 Significance of measles virus antibody findings 5 Summary References The failure to isolate virus from multiple sclerosis (MS) patients by cultural methods or by animal inoculation, or to identify by electron microscopy particles specific for the disease (Johnson, 1975), has increased the importance of the study of virus antibodies, in the hope that the immunological characteristics might point towards the causative agent. Since surveys of virus antibodies in serum have recently been fully discussed (Fraser, 1975), emphasis in this review will be given to reports that have led to the intensive investigation of measles antibodies in both serum and cerebrospinal fluid (CSF). Further investigations, in relation to measles and other antibodies, which require to be made, will be discussed. 1
Serological Surreys of Virus-Specific Antibodies in Multiple Sclerosis
Much effort has been expended on surveys of virus-specific antibodies in the sera of patients with MS and in those of controls to determine whether different responses in the groups might indicate that a virus or several viruses are involved in the pathogenesis of the disease. This has stemmed from the initial finding by Adams & Imagawa in 1962 that measles antibody titres were increased in MS patients compared with controls; subsequent reports, reviewed by Brody et al. (1972) and more recently by Fraser (1975), have supported this observation. In a discussion of 27 reported studies, using different laboratory techniques, Fraser (1975) noted significantly higher levels of measles antibody of the order of a 2-2$-fold excess in MS patients over controls. From our own studies (Haire et al. 1973a, b) and thereportsof many others, the scatter of individual titres has been shown to be small, and the antibody titre in each patient has not been shown to be of any diagnostic significance or to bear anyrelation to the clinical activity ofthe disease. In Sweden and Finland, workers have made an important contribution by their use of different measles antigens, and they have found significantly higher antibody titres to the three separate structural components, nucleocapsid, haemagglutinin and haemolysin (Salmi et al. 1972a; Salmi et al. 1973) in MS patients compared with healthy persons resident in the same 40 Downloaded from https://academic.oup.com/bmb/article-abstract/33/1/40/299852 by Insead user on 14 August 2018
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SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire used in the survey and MS, a conclusion already made by Nikoskelainen et al. (1972). Autoantibodies are commonly associated with virus infections (Holborow, 1972), but we have been unable to correlate them with virus antibodies. We have shown an inCanine distemper creased incidence and higher titres of virus smooth-muscle antibody (SMA),"fibrillar'* antibody to HEp 2 tissue-culture cells and 20.1 to cultured rat cerebellar cells, in the 11.9 IgM class, in 11 MS sera lacking measles 15.2 IgM, compared with age- and sex-matched normal control sera, while there was no difference in the IgG class. Antibodies detected by the three antigens did not correspond, though absorption with both human uterus and normal brain powderreducedthe titres considerably (PlateTV: C). The specificities are unknown, but the SMA IgM autoantibody may be evidence of active cell membrane changes in this group of patients, all clinically free of secondary complications such as infection, and is similar to that described in infectious and malignant diseases by Holborow (1972). Further work is necessary to test a larger series of MS sera including those with measles IgM, searching especially for antibodies to defined central nervous system (CNS) antigens. The only report of IgM antibody activity in MS sera, of which 1 am aware, is that of Dowling et al. (1968) who have shown 19 S and 7 S demyelinating activity in MS sera on cultured neonatal mouse cerebellum. Areviewof antibodies in MS sera would be incomplete without reference to neutralizing antibodies to the MS-associated agent (MSAA) (Henle et al. 1975). Carp et al. (1972) first described the suppression of polymorphonuclear neutrophils (PMN) in mice following inoculation of material from MS patients, and Henle et al. (1975) have confirmed that there is a transmissible MSAA and have extended the work to include serological evidence. The antibodies, of IgG class, are found in patients' sera, in sera of theirrelativesand of nursing personnel and in a considerable proportion of sera from East Africa, where MS is unknown. Sera from healthy American blood donors and from patients with virus infections seldom have such antibodies.
TABLE I. Geometric mean titres of antibody* to measles, rinderpest and canine distemper viruses in eight multiple sclerosis (MS) sera and five acute measles sera Number and type of sera
Immunoglobulin
Antibody to Measles virus
Rinderpest virus
MS (8)
IgG IgM
56.9 26.1
23.9 4.2
Acute measles (5)
IgM
34.8
10.0
* Titres of antibody are expressed as reciprocals
of MS sera at a rate similar to that which we reported in 1973 (Haire et al. 1973a,b). The antigen responsible for measles IgM activity in MS differs from that found in acute measles infection (Fraser et al. 1974) and its specificity is being investigated. In one investigation in collaboration with F Brown (Haire et al. 1974a), eight MS sera andfivesera obtained early in uncomplicated measles infection (all possessing IgM activity to the membranes of measles-virus-infected cells) were tested for both IgM and IgG antibodies, using cells infected with the cross-reacting viruses, rinderpest and canine distemper (Imagawa, 1968). With both groups of sera the titres against measles were highest (Table I), which suggests that in MS patients the antigenic stimulus is measles virus or one of its components rather than the other two related viruses. Our own and other reported findings of raised titres of antibody to herpesviruses in MS patients prompted us to make a serological survey of IgG and IgM antibodies to cytomegalovirus, varicella zoster, herpes simplex and EB viruses. No significant difference was found in the mean titres of the IgG antibodies between the MS patients and the two control groups (Table II). While neither EB IgM nor cytomegalovirus IgM was found, several sera had low titre IgM to one or both of the other viruses with equal distribution in the three study groups. As both herpes simplex and varicella zoster are latent viruses for man, we did not attach any importance to thisfinding,which confirmed an earlier observation regarding herpes simplex IgM (Millar et al. 1971). From our results we decided that there was no obvious connexion between the herpesviruses
2 Virus-Specific Antibodies in Cerebrospinal Fluid of Multiple Sclerosis Patients Studies of virus antibodies in CSF of MS patients have been restricted by limited quantities obtainable from patients, by the lack of suitable control specimens and by insensitive techniques not capable of detecting the small amount of antibody which might be present. Despite these limitations certain important studies have been reported during the pastfiveyears and are summarized in Table m . Brown et al. (1971) in their series also tested 65 MS CSF and 35 of the control CSF for antibodies to other myxoviruses including influenza A and B, parainfluenza 1-4, respiratory syncytial, rubella and mumps viruses, and while a few of thefluidshad a low titre of antibody, the outstanding finding was the high incidence and significantly highertitresof measles antibody detected by the sensitive mixed haemadsorbing antibody test. In 33 MS and 17 control patients, for whom matching CSF and serum specimens were available, statistical analysis did not show a significant over-all correlation between
TABLE II. Geometric mean titres of virus-specific IgG antibody* in patients with MS, normal control subjects and neurological control patients Virus
Herpes simplex Varicella zoster Cytomegalovl rus EB virus Number
Group MS patlentsf
Normal subjects!
Neurological controls^
45.8 21.9 9.3 31.4
31.9 11.9 11.3 24.1
53.9 15.5 18.5 33.3
39
39
35
* Titres of antibody are expressed as reciprocals t Age- and sex-matched subjects (16 males, 23 females) X Slightly older controls (16 males, 19 females)
41 Vol. 33 No. 1
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SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire CSF and serum antibody levels. Salmi et al. (1972b) showed that the one gel precipitation line produced by CSF from 2/15 MS patients against measles antigen had a reaction of identity with one of several lines produced by the CSF of a patient with subacute sclerosing panencephalitis (SSPE), in which condition measles antigen has been found in brain and extremely high titres of measles antibody have been measured in CSF
(Connolly et al. 1968). Complement-fixing (CF) antibody to 27 other viruses tested was not found in these two CSF. During 1973 it appeared possible that vaccinia virus, used for immunization against smallpox, might be involved in some way in the aetiology of MS, after Kempe et al. (1973) found neutralizing antibody to vaccinia in 30% of MS CSF, with less than 3 % in CSF of the neurological control group and none in a normal control group (medical students). In order to confirm this finding, Brown et al. (1973) tested CSF from 127 TABLE III. Studies of virus antibodies in cerebrospinal fluid (CSF) MS patients in France and from 78 of MS patients patients with other neurological disorders, and detected vaccinia antibody in none of Virus Antibody Test positive in Comment the MS patients and in one patient in the control group. Exchange of specimens beMS Neurological Normal controls controls tween the two laboratories ensured that there were no technical differences. One Mixed Measles* 94/119(79%) 51/112(45%) Increased frequency In possible explanation for the finding was MS; significantly higher haemadsorbing titres in MS that in France, where vaccination is per_ Measles* Gel predpitating 2/15(13%) Gel precipitation lines formed at 12 years of age without subwere identical with 1/3 sequent boosters, the chance of detectable lines produced by subacute scteroslng antibody in serum at the time of obtaining panencephalitis CSF CSF from MS patients would be minimal. Vacdnia Neutralizing 56/187(30%) 3/87 (3%) USA population which 0/30 had received fairly Kempe's specimens were from patients in extenslvo vaccination* the USA where, on the other hand, until Vacdnia Neutralizing 0/127 1/78(1%) French population which received vaccination at very recently, fairly extensive and frequent 12 years; boosters vaccination was carried out; so it is reasonrare* Paralnfluenxa Neutralizing 0/62 0/36 able to assume that serum antibody levels Negative findings type 1* would be high and that the CSF antibodies Measles* /•I6/25 (64%) 0/17 \ Measles, herpes simplex may be due to leakage across the bloodHerpes simplex* I 9/25(36%) 0/171 FAt IgG and mumps IgM was Mumps* i 0/25 0/17/ brain barrier. Brown et al. (1973) also tested not found in CSF Rubella* I 0/11 0/I6J 62 MS and 36 control CSF of this series for 19/301 (63%) I/30II.111 Measles 1/30" (3%) [. 9/19—measles IgM in serum neutralizing antibody to parainfluenza type li. Patient with jreneral 1 virus, with completely negative results. paralysis of the in«n«—measles IgM in serum In Belfast we have not found IgM antilil. Patient with brain body in CSF, of either MS patients or tumour and FAt IgG morbilliform rash control subjects, to any viruses we have iv. "Normal" control found to have tested, viz. measles, mumps, herpes simplex spastic paraparesls and rubella. This is not surprising, as Link •f extensor plantar & Muller (1971) did not show an increase responses Herpes simplex " 11/30(37%) 7/30 (23%) 2/30(7%) I 6/18(33%) Rubella' j 2/21 (9%) in total IgM in 64 MS CSF, though their 1/16(6%) Measles* 13/30 (43%) 2/30(7%) Haemagglutinin 2/30(7%) -v technique did reveal an increase in 9% of inhibiting (CSF and serum] CSF of 39 patients with infections of the Nudeocapsid Reduced serum: CSF 10/30 (33%) 0/30 0/30 complement ratios in 12/30 MS CNS. By indirect immunofluorescence we fixing patients; none In either HaemoTysln have found IgG antibody specific for 14/19(74%) 2/18(11%) 3/19 (16%) J control group inhibiting measles, herpes simplex and rubella viruses Measles* 23/80(29%) 1/97(1%) Haemagglutinin (Haire et al. 1973a,b) in the CSF of MS Inhibiting 0/98 Ribonudeoprotein 16/80(20%) Measles and rubella antipatients, but that to measles predominated gel predpitating bodies In positive cor1 relation with IgG and Haemolysin and in several separate studies the preval41/79(52%) 6/92 (6%) Inhibiting - ( higher ratio of ence has been consistent (Haire et al. 1974b). Haemaggtutlnin Rubella* 2/94(2%) lgG:albumin 18/84(21%) inhibiting We have tested CSF and serum obtained at Measles — ( 57%S the same time from 30 MS patients, 30 l9 Rubella Local production in CNS patients with other neurological diseases 1 7i Mumps Reduced _ to any of J '^%¥ and 30 " normal" controls who were patients - 1 oftheantibodies Herpes simplex serum:CSF ratiot i 1 l%j — viruses in 7 1 % MS - r patients type \J undergoing myelography for suspected proParainfluenza — 1 3%S type 1^ lapsed intervertebral disk, but we did not establish a positive correlation between the * Laboratory exchange of sped m e n checked that the results of Kempe et of. (1973) and of Brown et of. (1973) presence of measles IgG in CSF and were correct measles IgM in serum in MS (Haire et al. PA: Indirect Immunofiuorescence for IgG antibody Several different serological tests were employed 1973b), though it is interesting that 9/11 150 patients were studied patients whose sera had measles IgM had References: also measles IgG in CSF. In this series, o: Brown et of. 1971 d: Brown et of. 1973 g: Norrby et al. ]974a b: Salmi et of. 1972b e: Haire et al. 1973a Salmi et at. 1974 CSF from 10/30 patients had IgG to both c: Kemp* et of. 1973 f: Haire et al. 1973b / : Norrby et of. 1974b 0
4
/
7
7
7
7
- 1 -I
i
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Br. Med. Bull. 1977
PLATE III
SIGNIFICANCE OF VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS Margaret Haire (FIG. A-C)
A. "Fibrillar" staining pattern seen when fixed tissue-culture cells are treated with MS serum and anti-human IgM conjugated with fluorescein isothiocyanate (FITC) (mif nificition x 550)
B. Membrane staining of measles-vlrus-infected tissue-culture cells, seen when live infected cells are treated with MS serum and antihuman IgM conjugated with FITC (mainificacion x 550)
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PLATE IV
SIGNIFICANCE OF VIRUS ANTIBODIES IN MULTIPLE SCLEROSIS (continued)
CON
CON
MS
MS
CON
ill dii)
di)
MS
CON
MS
CON
-1 (ii)
(i)
c
MS •
CON
(iii) CON
MS
CON
MS
log,
o
>
-2-_
S
:
ant
-9 o
IILI.
• 1
Ill
H
1m (i)
ill (ii)
• •
(iii)
C. Titres of IgM antibody in 11 MS sera and In sera of age- and sex-matched controls ( C O N ) before absorption (I), after absorption with human uterus (II), and after absorption with normal brain powder (iii). Antigens used are: ( a ) composite blocks of rat liver, stomach and kidney to detect smooth-muscle antibody; ( b ) HEp 2 tissue-culture cells; and (c) cultured cells from cerebellum of newborn rats. Cultured cells are acetone fixed
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SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire neutralizing activity against the MSAA, whilefluidsfrom two control patients had no such effect. Two of three sera tested from these four MS patients also contained neutralizing activity. Information regarding autoantibodies in CSF is scanty. While Laurell & Link (1972) showed CF antibodies reacting with crude MS brain in 6/12 CSF from MS patients and in 0/17 CSF from neurological control patients, they also showed CF antibodies detectable at a lower titre in 5/6 CSF when normal brain antigen was used. As in the case of sera, further work is needed to detect antibodies to defined CNS antigens in CSF of MS patients, and to relate detectable antibody to the several membrane viruses and the MSAA which have already been shown to possess antibody activity within the CNS.
measles and herpes simplex viruses, and serum: CSF ratios of antibody in these 10 patients indicated lower ratios in the case of measles virus; but we are not prepared to conclude that this means active growth of virus in the CNS (Fraser, 1975). Clarke et al. (1965) found ratios of serum:CSF poliovirus antibody to be lower in MS patients compared with controls; and indeed this reduced ratio tended to be associated with higher globulin concentrations in the CSF, indicating that in MS at least some of they-globulin in CSF is derived from serum antibody. The report by Connolly et al. (1968) of significantly reduced ratios of titres of serum:CSF measles antibody in the three patients with SSPE, in whose brains measles antigen was found by immunofluorescence, prompted Norrby et al. (1974a) to use this method to seek evidence for measles antibody production in the CNS of MS patients. They compared the ratios of serum:CSF antibodies to different measles virus components with the ratios to two reference viruses, poliovirus and adenovirus, both unlikely to be involved in CNS disease. The study groups were carefuily selected; the CSF of 30 MS patients had two or more extra y-globulin bands (oligoclonal IgG) and the serum electrophoretic pattern was normal. Twenty-six of these patients had raised relative IgG concentration in CSF, while the IgG content of the CSF of the 30 neurological control patients was normal, though 26 of these 30 patients showed a slight to moderate increase in CSF total protein, suggesting damage to the bloodrbrain barrier. The "healthy" control group consisted of 30 patients with minor psychiatric disorders, without apparent CSF abnormality. Twelve of the 30 MS patients showed a significantly reduced ratio of serum: CSF antibody to measles haemagglutinin and measles nucleocapsid, with normal ratios in most cases to the two reference antigens. By measurement of antibody to measles haemolysin 5/19 MS patients studied also showed a reduction. None of the 60 control patients had this reduction, and, as the patients with neurological disorders had moderate damage to the blood-brain barrier, Norrby et al. (1974a) decided that the reduced ratios in MS are not due to selective passage of antibodies to the CNS, but rather to active production within the CNS. Most of the studies from the Scandinavian workers have been concerned with CSF antibodies to measles virus, but two later reports focus attention on rubella and other viruses. In Finland, Salmi et al. (1974) found antibodies in CSF to measles haemagglutinin, measles haemolysin, measles ribonucleoprotcin and rubella haemagglutinin in 28%, 51%, 20% and 20% of MS patients, respectively, with very few of the neurological control patients having any detectable antibody. Both measles and rubella antibodies were in positive correlation with higher levels of IgG and elevated IgG: albumin ratios in CSF specimens, suggesting that in MS some of these antibodies are synthesized within the CNS. A report based on a combined study of 150 MS patients from Sweden, Finland and Norway showed reduced ratios of titres of serum:CSF antibody, interpreted as indicating local production in the CNS, of antibody against measles virus in 57 %, against rubella virus in 19 %, against mumps virus in 15 %, against herpes simplex type 1 virus in 11 % and against parainfluenzatypelvirusin3%. Seventy-one per cent of them showed evidence of local production of antibody in the CNS against at least one virus (Norrby et al. 1974b). Henle et al. (1975) have shown that CSF from four patients with MS whose sera induced depression of PMN in experimental animals, implying the presence of the MSAA, had strong
3 Relation between Cerebrospinal Fluid Antibodies and Imnumoglobulins The presence of oligoclonal IgG in CSF and its absence from serum is very important in diagnosis in MS (Link, 1967), and several of the features of oligoclonal IgG may also give clues in the search for a cause of the disease. The location in both CSF and in MS brains (link, 1972) and the constancy of the band pattern during the course of the disease (Olsson & Link, 1973) may indicate that a responsible antigen, present in the CNS, is continually active. All attempts to investigate its specificity have been restricted to the detection of measles antibody in these bands. In one patient, Panelius & Salmi (1973) related both measles antihaemagglutinin (HI) and CF antibody with one of four bands, and HI antibody with another. Neither antibody was found in the other two bands, and tests were not carried out for antibody to different viruses. Vandvik & Degr6 (1975) found that one or two of several (up to eight) bands were associated with measles gel-precipitating antibody, and they speculated that the non-reactive bands may be antibodies directed against other virus components. Norrby & Vandvik (1975) succeeded, in the case of SSPE, by repeated absorptions with highly concentrated and purified measles virus, in removing almost completely all the oligoclonal IgG and in demonstrating that different bands showed antibody to different measles components. However, using a similar type of absorption of MS CSF they found it possible only in exceptional cases to alter the band pattern. Therefore, the presumed antibody activity of the major part of the oligoclonal IgG has still to be accounted for. Determination of the specificity of this antibody is essential in order to find an antigen which is very likely to be closely connected with the immunopathogenesis of the disease. 4 Significance of Measles Virus Antibody Findings The various surveys of serum antibodies show clearly that MS patients have a hyperactivity to measles virus, and reaction with three separate structural components of this virus (Salmi et al. 1972a; Salmi et al. 1973) adds strength to the view that measles virus may in some way be involved in the pathogenesis of the disease. Measles IgM, found by two groups of workers (Haire et al. 1973a,b; Karaseva et al. 1974), points to a continuing antigenic stimulus; and the greater IgM response in patients to measles rather than to the other viruses of the group, canine distemper and rinderpest viruses, further 43
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VoU33 No. 1
SIGNIFICANCE OF VIRUS ANTIBODIES Margaret Haire implicates measles in some way. Identification of the particular antigen involved is necessary, as it differs from that of a regular measles infection (Fraser et al. 1974), and until measles virus is shown in biopsy material from patients, in circulating leucocytes or in autopsy material, analysis of serum antibodies or search for immune complexes are the only feasible methods of achieving this. One clue may be that the IgM SMA may be stimulated by an immunologically active membrane antigen, resulting from virus-membrane interaction in the patient While antibody to measles virus predominates in CSF in MS patients, Norrby & Vandvik (1975) did not find a clear correlation between bands of oligoclonal IgG and measlesvirus-specific activity in all patients. In order to explain these bands, for whose presence a responsible antigen or antigens have not been found, it would be worth searching for activity in them to rubella, mumps and herpes simplex viruses, all of which have been shown to produce antibody in CSF of MS patients (Haire et al. 1973b; Norrby et al. 1974b); activity to defined CNS antigens should also be investigated. Assessment of antibody to the MSAA, in both serum and CSF, will be of real value only when a simpler and more reproducible method of assay has been found. If a better assay is developed, full evaluation of the role of MSAA in MS will
be necessary. First, this agent may cross-react with measles, causing increased antibody levels in patients. Secondly, it may affect the cellular immune mechanisms, with resulting elevation of humoral antibody to measles, or, thirdly, it may be an independent infectious agent in some patients. 5 SuDsroary Surveys of virus-specific antibodies in sera and CSF of MS patients give evidence that measles virus may be involved in pathogenesis. The specificity of antibody activity of oligoclonal IgG in CSF will give more precise knowledge of antigens active in MS. ACKNOWLEDGEMENTS
I wish to thank D r J H D Millar, Dr Michael Swallow and Dr J A Lyttle, who have made clinical specimens available. The work reported here has been carried out with the assistance of Dr W M Hutchinson, Mrs Alison McKee, Mrs Patricia McCullough, Mrs Ann Simpson, Miss Caroline Simpson and Mr S A McMillan. Mr McMillan kindly supplied thefiguresfor Plates HI, IV. Research work in multiple sclerosis in the Department of Microbiology and Immunobiology, The Queen's University of Belfast, is supported by a Programme Grant from the Medical Research Council.
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