Cerebrospinal Fluid and Serum Immunoglobulins and

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1 INFECTION AND IMMUNITY, Sept. 1978, p /78/21-852$2./ Copyright 1978 American Society for Microbiology Vol. 21, No.3 Printed in U.S.A. Cerebrospinal Fluid and Serum Immunoglobulins and Antibody Titers in Mumps Meningitis and Aseptic Meningitis of Other Etiology A. FRYDgN,'* H. LINK,2 AND E. NORRBY3 Departments of Infectious Diseases' and Neurology,2 University Hospital, Linkoping, and Department of Virology,3 Karolinska Institutet, Stockholm, Sweden Received for publication 13 June 1978 Cerebrospinal fluid (CSF) and serum from 19 patients with mumps meningitis and 19 patients with meningitis of other etiology were investigated on two or more occasions for at least 1 month after onset. Intrathecal synthesis of immunoglobulin (Ig) G was found in 55%, of IgA in 26%, and of IgM in 2% of the patients. Oligoclonal Ig was demonstrable by agarose gel electrophoresis in 37% of the patients, mostly already during the first week after onset, and could persist for years. Mumps virus antibody synthesis within the central nervous system occurred in 37% of the mumps meningitis patients. The inflammatory reaction within the central nervous system as reflected by mononuclear pleocytosis, Ig synthesis, and oligoclonal Ig was not correlated to the clinical course. The bloodbrain barrier was evaluated by determination of the CSF total protein, CSF/serum albumin ratio, and CSF/serum a2-macroglobulin ratio. A significant correlation was found among these three parameters. Persistence of the elevated CSF/serum albumin ratio seems to influence prognosis, and this parameter is recommended for evaluation of the blood-brain barrier function. Evidence has accumulated indicating that the central nervous system (CNS), from an immunological point' of view, must be considered partly as a separate entity. Thus, in the case of mumps meningitis, increased T and decreased B lymphocyte populations have been found in cerebrospinal fluid (CSF) as compared to blood (3). Decreased responses to mitogens (, 1), but increased responses to mumps virus on stimulation of CSF, compared to blood lymphocytes, have also been observed in mumps meningitis patients (5). These different immunological reactions of CSF in comparison with blood lymphocytes occur although the CSF lymphocytes have been shown to derive from blood (25). Elevated immunoglobulin (Ig) levels in CSF, partly due to synthesis within the CNS, and intrathecally synthesized oligoclonal Ig have been demonstrated in aseptic meningitis (1). The importance of the cell-mediated immune response in viral CNS infection is experimentally documented, whereas the significance of the humoral immune response is not yet clarified (6, 7). No investigation has hitherto been published in which the Ig in CSF and serum have been analyzed in consecutive samples during the course of aseptic meningitis. Thus, the frequency of Ig abnormalities and occurrence of oligoclonal 852 CSF Ig in aseptic meningitis is not known, nor is the time of appearance and the persistence of these abnormalities. The aim of the present paper is to investigate the occurrence of Ig abnormalities in the form of elevated Ig levels and of locally synthesized polyclonal or oligoclonal Ig in CNS during the acute phase and convalescence of aseptic meningitis caused by mumps virus and, for comparison, by other infectious agents. In the patients with mumps meningitis, antibody titers against mumps virus antigen were determined in parallel, and variations in CSF and serum titers were correlated to Ig alterations. The blood-brain barrier function was investigated by determination of the CSF total protein and of the CSF/serum ratios of albumin and of a2-macroglobulin. These laboratory parameters were correlated to severity of the disease. MATERIALS AND METHODS Thirty-eight patients with acute aseptic meningitis treated at the Department of Infectious Diseases were studied. Most patients were treated during the hospital stay and then followed up by one of us (A.F.). The diagnosis was based on clinical findings, leukocytes in CSF exceeding 5 x 16/liter in 37 of the 38 patients, and the course of the disease with recovery without antibiotic treatment. The patients were divided into two groups. Group

2 VOL. 21, 1978 A included 19 patients 2 to years old (mean, 33 years) with serologically confirmed mumps meningitis. Group B also included 19 patients, 16 to 6 years old (mean, 32 years). In this group the etiology of meningitis could be established with certainty in six patients (individual cases caused by herpes simplex type 1, varicella-zoster, ECHO 3, influenza A, Coxsackie B, and tick-borne encephalitis virus), while a probable etiological agent was detected in four additional patients (Mycoplasma pneumoniae in 3 and enterovirus in 1). The etiology remained unknown in the remaining nine patients. All patients were above 15 years old and were informed of the purpose of the study. Only those who gave full consent were included. CSF and serum were taken at the same time from each patient on three occasions, namely on the day of admission (specimen I), about 5 days after admission (specimen II), and 3 to 6 days after admission (specimen III). Because the interval between onset of meningitis symptoms and admission was rather constant ( to 5 days; mean, 2 days), the day of admission was accepted as the starting point for the investigation. Additional CSF and serum specimens (specimen IV) were obtained more than 3 months after onset from those patients who displayed CSF abnormalities in specimen III and/or persisting symptoms. Cell counts and determinations of total protein, IgG, and albumin were carried out immediately after lumbar puncture on all CSF specimens. The erythrocyte count in CSF did not exceed 1, x 16/liter and was 1 x 1" to 82 x 16/liter in 12 of the CSF specimens. IgG and albumin were determined simultaneously in the CSF and in the serum, and the CSF-IgG index was determined. The specimens were then stored at -21C. A portion of the CSF was concentrated by ultrafiltration in collodium bags (Sartorius Membranfilter, Gottingen, W. Germany) at C to an IgG concentration of about 3 g/liter and then analyzed by agarose gel electrophoresis in parallel with serum. All specimens from individual patients were then investigated simultaneously regarding IgA, IgM, kappa and lambda light chains, a2-macroglobulin, and complement factors C3 6ic/fi, globulin) and C (,61E globulin). Immunochemistry. After cell counting was performed by phase-contrast microscopy, the CSF was centrifuged. The total protein concentration in CSF was determined according to Lowry et al. (19). The age-dependent reference values calculated by Tibbling et al. (32) were adopted (Table 1). CSF Ig VARIATIONS IN MENINGITIS 853 Determinations of albumin, IgG, IgA, and kappa and lambda light chains were carried out on unconcentrated CSF and on serum by an automatic immunoprecipitation technique utilizing nephelometric analyses of antigen-antibody complexes in a continuous flow system (Auto-analyzer II, Technicon Corp., Inc., Tarrytown, N.Y.). a2-macroglobulin, IgM, and the complement factors C3 and C were determined by single radial immunodiffusion (2), modified as described (18). Antisera against albumin, IgG, IgA a2-macroglobulin, and kappa and lambda light chains were purchased from Dakopatts (Copenhagen, Denmark), antisera against IgM and C3 was from Organon Teknika (Oss, Holland), and antiserum against C was from Behringwerke (Marburg-Lahn, W. Germany). The reference values for the individual proteins in CSF and serum are given in Table 1. These values refer to a normal material defined according to Tibbling et al. (32), consisting of 56 individuals with functional neurological disorders but normal findings at neurological examination and with normal serum proteins. The CSF/serum albumin ratio and the CSF total protein increase with age, the upper normal values being 5.7 and 3 mg/liter, respectively, at 3 years, and 9.7 and 658 mg/liter, respectively, at 6 years (32). The IgG level in CSF was presented by the CSF- IgG index equal to (CSF IgG/serum IgG):(CSF albumin/serum albumin) (32). The CSF-IgG index takes into account the influence of fluctuations of serum IgG and of blood-brain barrier disturbances (15). An elevated CSF-IgG index value indicates the occurrence of IgG synthesis within the CNS (16). The IgA and IgM concentrations in CSF were similarity presented by the CSF-IgA and CSF-IgM indexes, respectively. The upper normal values in our laboratory are.7 for the CSF-IgG index, and.62 for the CSF-IgA index. The lower limit of IgM detected with the method used was 2 mg/liter, and IgM was therefore not demonstrable in normal unconcentrated CSF. Although a normal range for the CSF-IgM index has not been defined as accurately as it has for the other Ig's, >.6 appears to be a raised value. We only evaluated >1. as an abnormal value. The kappa/lambda ratio (17) was determined. The 95% confidence limits in our laboratory are.7 to 1.7 for CSF, and.7 to 1.3 for serum. Agarose gel (Behringwerke) electrophoresis was carried out as described previously (13). The occurrence of one or more homogeneous bands in the TABLE 1. Reference values for CSF and serum proteins given as 95% confidence limits Protein CSF (mg/liter) Serum (g/liter) CSF/serum ratio Indexa Total protein' Albumin" < IgG IgA IgM < '1. a2-macroglobulin C C For explanation see text. b The normal values are taken from Tibbling et al. (32).

3 85 FRYDI8N, LINK, AND NORRBY gamma globulin region in addition to those normally seen was considered to be abnormal. Viral serology. The following four tests were used for determination of mumps virus antibodies in serum and CSF: hemagglutination inhibition, complement fixation, hemolysis inhibition, and mixed hemadsorption. Details of the techniques used were described previously (21). Hemagglutination-enhancing antibodies against adenovirus were also determined (2). A local production of mumps virus antibodies within the CNS was concluded to occur when the ratio of serum/csf antibody titers was at least four times lower than the corresponding ratio of adenovirus hemagglutination-enhancing antibody titers. Statistical methods. Student's t test was used. The correlation coefficients were calculated as ordinary product-moment coefficients. RESULTS Pleocytosis. All but one of the 38 patients with aseptic meningitis displayed pleocytosis, which varied during different phases of the disease (Fig. 1). A pleocytosis was still demonstrable in specimen III at more than 1 month after onset of meningitis in 11 of the 18 patients investigated in group A (61%) and in 11 of the 19 patients in group B (58%). In specimen IV, when lumbar puncture was repeated more than.1 3 CSF Ieiuocytes A 2 i 2. I I III IV icsf suiwtes I II ~III IV SpcLima Nube FIG. 1. Numbers ofpatients belonging to group A (A) and group B (B) with pleocytosis (filled bars) in CSF specimens obtained at different times after onset ofsymptoms. Unfilled bars represent the total number ofpatients investigated. B INFECT. IMMUN. 3 months after onset of meningitis symptoms, the pleocytosis had disappeared in all seven mumps meningitis patients examined, but was still present in two of the four patients belonging to group B which were studied. Polynuclear cells were observed in CSF specimen I in about 2/3 of the patients and in about 1/3 in specimen II, but never thereafter. Blood-brain barrier damage. Evidence of blood-brain barrier damage as documented by elevated CSF/serum albumin ratios was obtained in 69% of specimen I, 53% of specimen II, and 32% of specimen III (Fig. 2). The corresponding figures for elevated total protein were 76, 81, and 1%, respectively, and for elevated CSF/serum a2-macroglobulin ratio were 2, 28, and 13%, respectively. The correlation coefficients (r) between these three parameters are given in Table 2. CSF and serum Ig's. Altogether, 13 of the patients belonging to group A and 8 of the patients in group B displayed elevated CSF-IgG index values at some time during the course of the disease. Elevated values were found in 11 out of 32 CSF specimens I (3%), and in 11 out of 29 CSF specimens II (38%) (Fig. 3). The CSF- IgG index had become normalized in 9 out of the 11 patients with elevated values in specimen I. Eight patients with normal values in specimen I displayed abnormally high values in specimen II. Nine patients displayed elevated CSF-IgG index values, two of them for the first time, in specimen III. Finally, 7 out of 15 patients (7%) displayed elevated CSF-IgG index values in specimen IV. Elevated CSF-IgA index values were found only infrequently, mainly in specimen III (16%) (Fig. 3). Three of the five patients with elevated values in specimen III had normal values in the two previous specimens. Elevated CSF-IgM index values were found in nine patients (Fig. 3). Two patients (7%) had elevated values in specimen I, four (16%, three of them for the first time) in specimen II, and five (17%, four of them for the first time) in speci- -n III. Only one of the patients with an elevated CSF-IgG index or CSF-IgA index, and no patient with an elevated CSF-IgM index, had elevated serum levels of the corresponding Ig. Oligoclonal 1g. The presence of oligoclonal Ig in CSF but not in serum, indicating Ig synthesis within the CNS, was demonstrated by agarose gel electrophoresis in 7 of the 19 patients in each group (37%). Five of the seven patients belonging to group A displayed oligoclonal CSF Ig in specimen I, and the remaining two patients displayed it first in specimen III. All seven patients belonging to group B had oligoclonal CSF

4 VOL. 21, 1978 CSF Ig VARIATIONS IN MENINGITIS 855 cc z I D I cc Uj 2 Uc CLtC f o... I II SPECIMEN SPECIMEN III NUMBER. * *1. NUMBER IV *t A* cc -j z m co 2C I n 5t 3,*.8 1* I i *to re r * i " ~~~: SPEIMNU SPECIMEN NUMBER es~~~~~~ IE FIG. 2. Blood-brain barrier function represented by CSF/serum albumin ratio, CSF total protein, and CSF/serum a2-macroglobulin ratio in patients with acute aseptic meningitis at different times after onset of symptoms. The normal values of CSF/serum albumin ratio and CSF total protein are age dependent. Line represents upper normal level of CSF/serum a2-macroglobulin ratio. Symbols:, Normal values; *, elevated values. Ig already in specimen I. Two additional patients, one with herpes zoster meningitis and the other with Coxsackie B meningitis, had oligoclonal Ig in CSF as well as in serum. The number of bands was higher and the intensity more pronounced in CSF compared to serum, indicating that oligoclonal Ig derived from the CNS. Lumbar punctures were repeated in some of the patients with oligoclonal CSF Ig. Among the seven patients belonging to group A, the oligoclonal CSF Ig had disappeared in one patient 1 year after the onset of symptoms. In the remaining six patients, oligoclonal Ig remained in late convalescence samples of CSF. In two of them, the last lumbar puncture was performed 1 to 2 months after onset, and in two additional patients the last puncture was 1 year after onset.

5 856 FRYD2N, LINK, AND NORRBY TABLE 2. Correlation coefficients and P values for protein parameters used for evaluation of blood-brain barrier function Ia II III Correlation between: rb P r P r P CSF/serum albumin ratio and CSF.5 <.1.95 <.1.37 <.5 total protein CSF/serum albumin ratio and.72 <.1.61 <.1.2 NSc CSF/serum a2-macroglobulin ratio CSF total protein and CSF/serum.52 <.1.58 <.1.98 NS a2-macroglobulin ratio a Specimen number. b r, Correlation coefficient. e NS, Not significant. It was not possible to obtain additional specimens from any of these four patients. In the remaining two patients with mumps meningitis, repeated lumbar punctures were carried out during 2.5 years after onset. Oligoclonal CSF Ig was detectable in all samples. Among the seven patients belonging to group B with oligoclonal CSF Ig, the bands had disappeared 1 to 2 months after onset in two of the patients, and 3 to 6 months after onset in two additional patients. In the three remaining patients, the oligoclonal Ig was still detectable 2 months after onset in one patient, and 6 months after onset in two patients. It was not possible to obtain additional CSF specimens. In those instances where multiple specimens were analyzed, the oligoclonal CSF Ig was mostly reduced both with regard to number of bands and intensity of staining during the time of observation. Thus, one patient displayed five oligoclonal Ig bands at onset and only two Ig bands 2.5 years later. On the other hand, in CSF from two patients the bands could be seen to increase from two to four during observation periods of 6 and 12 months, respectively. Additional evidence for the presence of oligoclonal Ig in CSF was obtained by the finding of an abnormally high kappa/lambda ratio of CSF in six patients belonging to group A (Table 3) and in five patients of group B. One additional patient displayed an abnormally low ratio. There was no clear-cut parallelism between the occurrence of oligoclonal CSF-Ig demonstrable by agarose gel electrophoresis and the finding of abnormal kappa/lambda ratios. Thus, abnormal ratios were found in 6 patients in whom agarose gel electrophoresis did not reveal oligoclonal Ig. However, all patients but one with abnormal kappa/lambda ratios displayed elevations of one or more CSF-Ig levels. The kappa/lambda ratios of serum were normal in all 38 patients. Mumps virus antibody synthesis within the CNS. Of the 38 patients, 37 had demonstrable mumps virus antibodies in their serum. INFECT. IMMUN. Mumps antibodies could also be detected in the CSF in 9 of the 19 patients of group A. Based on the ratio of CSF/serum mumps virus antibodies, evidence for a local synthesis in the CNS of these antibodies was obtained in seven of these nine patients. Of these seven patients, six had CSF-IgG index values above.9. The hemagglutination inhibition, hemolysis inhibition, and mixed hemadsorption tests detect antibodies against surface antigens only, whereas the complement fixation test detects, in addition, antibodies against the inner components. The complement fixation test is, however, insensitive and was positive in only one of seven patients in which mumps virus antibody synthesis was demonstrable, compared with 2/7 in hemolysis inhibition, 6/7 in hemagglutination inhibition and 7/7 in mixed hemadsorption tests. No mumps virus antibodies could be found in CSF in the remaining 1 patients with mumps meningitis or in the 18 patients of group B tested. When the 19 mumps meningitis patients were subgrouped according to presence or absence of oligoclonal CSF Ig demonstrable by agarose gel electrophoresis (Table 3), evidence for mumps virus antibody synthesis within the CNS was obtained in four patients with oligoclonal Ig and in three without this CSF abnormality. Regarding those three last-mentioned patients, the only abnormality was an elevated CSF-IgG index and CSF-IgM index in one patient each, indicating synthesis of the corresponding Ig within the CNS. Complement factors. Elevated CSF/serum ratios of C3 were found in 26 patients, and elevated ratios of C were found in 19. Seven patients, who all belonged to group A, had elevated CSF/serum ratios of C3 (four patients) or C (five patients) in the presence of a normal blood-brain barrier. Two of the patients displayed elevated values for C3 as well as C. The serum levels of C3 and C were normal in all these seven patients. Four of them also had oligoclonal CSF-Ig, and one additional patient

6 VOL. 21, 1978 CSF Ig VARIATIONS IN MENINGITIS V) u c 2.21 M. V) u 5 **^. Specimen Number 2 As l.b 15< 5 1 * ~~~~~~~~~~~~~~~~~~~~~v.::::: **o* :I Specimen Number S C.) 5 *. C Specimen.I Number FIG. 3. Values of CSF-IgG index, CSF-IgA index, and CSF-IgM index in patients with acute aseptic meningitis at different times after onset ofsymptoms. Lines represent upper normal levels. Symbols:, Normal values; *, elevated values. had evidence of mumps virus antibody synthesis within the CNS. No marked mononuclear pleocytosis was noticed in any of the seven patients (O to 9 x 16/liter). Abnormally low CSF/serum ratios of C3 or C were not registered. Correlation between clinical and laboratory parameters. The severity of aseptic meningitis was judged by the patient's length of hospital stay and time of working inability. There was no obvious difference in severity of disease between group A and group B (Fig. ). No correlation was found between severity of disease and magnitude of pleocytosis, bloodbrain barrier damage in the acute stage of the disease, and presence of oligoclonal Ig or mumps virus antibody production within the CNS. How-

7 858 FRYD9N, LINK, AND NORRBY TABLE 3. Relation between oligoclonal CSF Ig's demonstrabke by agarose gel ekectrophoresis, and other laboratory parameters in acute mumps meningitis Oligoclonal CSF Ig's CSF abnormality Present (n Absent (n =7) = 2) Elevated IgG index 7 5 Elevated IgA index 2 2 Elevated 1gM index 2 1 Abnormal kappa/lambda ratio 3 3 Mumps virus antibody synthe- 3 sis within the CNS oars WMki ingabityr I M M'k' FIG.. Numbers ofpatients belonging to group A (mumps meningitis) and group B (meningitis of other etiology) according to duration of hospital stay and working inability. ever, a significant correlation was found between duration of hospital stay (P <.5), time of working inability (P <.1), and blood-brain barrier damage measured as an elevated CSF/serum albumin ratio in specimen III. Elevated CSF-IgG index and oligoclonal CSF- Ig were found in some instances without a concomitant CSF pleocytosis, especially late in the course. In specimen IV, this was always true. In the acute stage, only one patient had an elevated CSF-IgG index but a normal CSF cell count. All seven mumps meningitis patients with oli- INFECT. IMMUN. goclonal CSF Ig demonstrable by agarose gel electrophoresis displayed elevated CSF-IgG index (Table 3). In group B, only one of the patients with oligoclonal. CSF Ig had a normal CSF-IgG index. This patient also displayed normal CSF-IgA and CSF-IgM index values. In five additional patients, slightly elevated CSF-IgG index values were found in the absence of oligoclonal CSF Ig demonstrated by agarose gel electrophoresis, as well as determination of CSF kappa/lambda ratios. DISCUSSION Our results show that a persistent pleocytosis in CSF during 1 to 2 months after onset of symptoms is not a rarity in acute aseptic meningitis and was found in 2 out of 35 patients investigated (57%) independently of the course of the disease. These findings are in accordance with earlier reports (1, 33). However, a pleocytosis may occur 1 to 2 months after onset of acute aseptic meningitis, without influencing prognosis. The CSF/serum albumin ratio has been proposed to be a better parameter for demonstration of blood-brain barrier damage compared to the total protein (15). The higher frequency of abnormal total protein values compared to CSF/serum albumin ratios may be partly explained by the frequent occurrence of Ig synthesis within the ONS. Increased influx of serum low-molecular-weight proteins through a damaged blood-brain barrier also contributes to elevated CSF total protein. The concentration of individual proteins in CSF seems to be influenced by the molecular weight and configuration of the proteins, favoring entrance of proteins with low molecular size (18) and small volume (2). The present findings of considerably lower frequencies of elevated CSF/serum a2-macroglobulin ratios compared to elevated CSF/serum albumin ratios can be explained by the difference in molecular size and configuration between these two proteins, and the CSF/serum a2-macroglobulin ratio is less suitable as a parameter for determination of the blood-brain barrier function. To our knowledge, no adequate investigations have previously been presented regarding the concentrations of IgG, IgA, and IgM in CSF in acute aseptic meningitis or regarding fluctuations of these Ig's during the -course of the disease. One study included other types of ONS infections (1), and in another study the influence of a damaged blood-brain barrier or of elevated serum Ig levels was not taken into account when the CSF Ig levels were presented (31).

8 VOL. 21, 1978 Elevated CSF-Ig index values indicate Ig synthesis within the CNS, since these index values take into account the influence of a damaged blood-brain barrier as well as of elevated serum Ig (15). The present data indicate that synthesis of IgG, IgA, and IgM may occur in the CNS in patients with acute aseptic meningitis. Sixteen of the patients in group I (8%) and 1 of the patients in group II (7%) displayed intrathecal synthesis of any of these Ig's. Evidence for Ig synthesis within the CNS was obtained as frequently in the acute phase of the disease, i.e., in specimen I, as in specimens II and III obtained up to 2 months after onset of the disease. Our finding of intrathecal Ig synthesis is corroborated by in vitro investigations, where CSF lymphocytes obtained from aseptic meningitis patients were capable of synthesizing IgG and IgA as well as IgM (3). In some instances evidence for Ig synthesis within the CNS was obtained without simultaneous occurrence of the CSF pleocytosis, and it may be assumed that cells within the meninges are responsible for Ig production in these cases. Oligoclonal Ig in CSF from patients with aseptic meningitis has been reported at frequencies between 19 and % (9, 11, 1). Our finding of oligoclonal Ig in CSF in 37% of the patients is thus consistent with these earlier reports. However, B. Vandvik, E. Norrby, J. Steen-Johnsen, and K. Stensvold (manuscript in preparation) could not identify oligoclonal Ig during the first week of mumps meningitis, which contrasts to our finding of oligoclonal Ig already during the first week. The appearance of oligoclonal Ig in CSF early in the disease may indicate that not only hyperimmunization and prolonged antigenic stimulation are responsible for the occurrence of oligoclonal Ig. A parallel can be drawn to multiple sclerosis (MS), where synthesis of oligoclonal IgG within the CNS occurs. In MS, genetic factors have been shown to be involved (8) İf the occurrence of abnormal CSF kappa/lambda ratio in addition to oligoclonal bands is also taken into account, about half of the patients in both groups had oligoclonal Ig. Abnormal ratios were registered during the first week after onset in four of the six patients where this abnormality was the only evidence for ohgoclonal CSF Ig. As in MS, the kappa/lambda ratio was mostly increased (1, 17). This contrasts to the finding in Guillain-Barre syndrome, where abnormally low ratios are found as evidence for synthesis of Ig containing mainly lambda light chains (H. Link, B. Wahren, and E. Norrby, manuscript in preparation). The presence of oligoclonal Ig in CSF but not CSF Ig VARIATIONS IN MENINGITIS 859 in serum indicates that Ig is synthesized within the CNS. In fact, the majority of patients with oligoclonal Ig displayed abnormally high CSF-Ig index levels. It may thus be anticipated that patients with acute aseptic meningitis may synthesize Ig of one or more classes as well as oligoclonal Ig during the disease. These abnormalities, however, do not have any significant influence on the course of the disease, and their significance in acute aseptic meningitis has to be clarified. Our findings indicate that not all patients with mumps meningitis and oligoclonal CSF Ig display evidence of mumps virus antibody synthesis within the CNS, as has also been reported by Vandvik et al. (manuscript in preparation) who observed that two out of their four mumps meningitis patients with oligoclonal CSF Ig had no detectable mumps virus antibody synthesis within the CNS. However, this finding does not contradict that the oligoclonal CSF Ig present in mumps meningitis consists of mumps virus antibodies. Antibodies may be directed against the inner components of the virion not detectable by the insensitive CF test. Also in this respect, a parallel may be drawn with MS, which is characterized by CSF abnormalities similar to those in aseptic meningitis, including oligoclonal IgG (1) and high antibody titers against different viruses (22, 23, 29). When measle virus antibodies were studied in MS CSF, those directed against the virus nucleoprotein were found more frequently and at higher titers than antibodies against the virus envelope (28). Mumps virus antibodies have been detected in CSF from about 15% of MS patients (23). In contrast, we could not demonstrate such antibodies in 1 of our 19 mumps meningitis patients nor in any of the 18 patients with aseptic meningitis due to other etiology tested. Among these 18 patients, 7 had oligoclonal CSF Ig. Therefore, it can be presumed that specific antibodies against different viruses as observed in MS CSF (23, 29) do not occur frequently in aseptic meningitis. The present finding of mumps virus antibody synthesis within the CNS in three patients who did not display oligoclonal CSF Ig by agarose gel electrophoresis (Table 3) or by determination of the CSF kappa/lambda ratio may be due to insensitivity of the immunochemical techniques used or that the mumps virus antibodies were polyclonal. Two of the three patients had raised IgG index values (.86 and.91, respectively), indicating IgG synthesis within the CNS. At least 9% of MS patients display oligoclonal IgG bands, the number and mobility of which are constant, although their intensity may vary

9 86 FRYD2N, LINK, AND NORRBY (26). Upon investigation of our patients with aseptic meningitis by agarose gel electrophoresis of up to six consecutive samples, the oligoclonal Ig bands disappeared one by one, but also the opposite occurred, namely that they increased in number. In both situations the patients were clinically healthy. The finding of oligoclonal CSF Ig up to 2.5 years after uncomplicated acute aseptic meningitis is of importance when the demonstration of this CSF abnormality is used for diagnostic purpose, e.g., in MS. Persistence of oligoclonal Ig may be due to a latent infection in the CNS or to an unspecific stimulation of the immune system resulting in continuous antibody synthesis within the CNS. The presence of oligoclonal CSF Ig in the two serious and chronic diseases MS and subacute sclerosing panencephalitis could arouse the suspicion that the occurrence of oligoclonal Ig in patients with aseptic meningitis could predict an unfavorable course of the disease. In fact, Lindbak et al. (11) also made observations in four patients favoring this hypothesis. However, we could not confirm this since our 1 patients with oligoclonal CSF Ig had a course without any acute or late neurological complications, as well as the same hospital stay and working inability as the aseptic meningitis patients without oligoclonal CSF Ig. The immunodiffusion technique used for complement quantitation measures both the active and the conversion products. Elevated ratios of CSF/serum C3 and C were common in aseptic meningitis, but depended mostly on transfer from serum. In some mumps meningitis patients, increased CSF/serum ratios of C3 and of C were registered in the presence of an intact blood-brain barrier, suggesting the possibility of complement activation and/or synthesis within the CNS. Elevated C3 and C levels in CSF have also been reported in MS (26), and in exacerbations of systemic lupus erythematosus (27), indicating the possibility that immunological reactions are important. The results obtained in this investigation further confirm the opinion that the CNS regarding humoral immunity should be considered as a separate immunological entity. However, the clinical importance of the immune reactions still remains to be elucidated. ACKNOWLEDGMEENTS We thank Monika Dufmats and Ingela Lilja for skillful technical assistance. This investigation was supported by grants from the Swedish Medical Research Council (project no. 3381) and the Swedish Multiple Sclerosis Society. LITERATURE CITED 1. Azimi, P. H., S. Shaban, M. D. Hilty, and R. E. INFECT. IMMUN. Haynes Mumps meningoencephalitis. Prolonged abnormality of cerebrospinal fluid. J. Am. Med. Assoc. 23: Felgenhauer, K Protein size and cerebrospinal fluid composition. Klin. Wschr. 52: Fryden, A B and T lymphocytes in blood and cerebrospinal fluid in aseptic meningitis. Scand. J. Immunol. 6: Fryden, A., and H. Link Mitogen stimulation of cerebrospinal fluid lymphocytes in aseptic meningitis. Acta Neurol. Scand. 57: Fryden, A., H. Link, and E. Molier Demonstration of CSF lymphocytes sensitized against virus antigens in mumps meningitis. Acta Neurol. Scand. 57: Hapel, A. J The protective role of thymusderived lymphocytes in arbovirus-induced meningoencephalitis. Scand. J. Immunol. : Hapel, A. J., and I. Gardner Appearance of cytotoxic T cells in cerebrospinal fluid of mice with ectromelia virus-induced meningitis. Scand. J. Immunol. 3: Jersild,., T. Fog, G. S. Hansen, M. Thomsen, A. Svejgaard, and B. Dupont Histocompatibility determinants in multiple sclerosis with special reference to clinical course. Lancet ii: Laterre, E. C., A. Callewaert, J. A. Heremans, and Z. 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