Anti-Borrelia burgdorferi Antibody Response over the Course of Lyme Neuroborreliosis

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1 INFECTION AND IMMUNITY, Mar. 1991, p /91/ $02.00/0 Copyright ) 1991, American Society for Microbiology Vol. 59, No. 3 Anti-Borrelia burgdorferi Antibody Response over the Course of Lyme Neuroborreliosis SHAHID BAIG,' TOMAS OLSSON,' KLAUS HANSEN,2 AND HANS LINKlt* Department of Neurology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden,1 and Borrelia Laboratory, Department of Treponematoses, Statens Seruminstitut, Copenhagen, Denmark2 Received 13 August 1990/Accepted 13 December 1990 Characteristic findings on examination of cerebrospinal fluid (CSF) in Lyme neuroborreliosis include mononuclear pleocytosis, oligoclonal immunoglobulin G (IgG) bands, and evidence for local production of specific antibodies. We utilized an immunospot assay to detect cells secreting anti-borrelia burgdorfjeri antibodies of different isotypes over the course of disease. Such cells were detected in CSF from 13 consecutive patients with neuroborreliosis examined before treatment. IgG antibody-secreting cells were present in high numbers (mean, 32 cells per 104 CSF cells), whereas IgA and IgM antibody-secreting cells were found less frequently and at lower numbers (mean, 5 and 6 cells per 104 CSF cells, respectively). Clinical improvement after penicillin treatment was paralleled by a rapid decline of antibody-secreting cells in CSF, but they were still detected, although at lower numbers, in 5 of 10 patients examined more than 6 months after treatment. This specific B-cell response persisted despite clinical improvement. Whether it reflects persistence of antigen is unsettled. Neurological signs and symptoms resulting from infection with the tick-borne spirochete Borrelia burgdorferi may vary from involvement of a single cranial nerve to severe damage to the central nervous system, such as spastic paraparesis (4, 7, 9, 27, 33, 35). Routine examination of the cerebrospinal fluid (CSF) in Lyme neuroborreliosis may show a mononuclear pleocytosis in the range of 100 to 300 cells per mm3 (24, 28). This seems to be a regular finding in stages 1 and 2 of the disease, but it may be absent in stage 3. The CSF/serum albumin ratio or total CSF protein is frequently increased, reflecting blood-brain barrier damage. Most patients have local immunoglobulin production as manifested by an elevated immunoglobulin G (IgG) index and oligoclonal IgG bands in CSF (15, 19, 25, 30). Intrathecal synthesis of IgA and/or IgM has also been reported (15, 18, 29). Lyme neuroborreliosis is accompanied by elevated titers of antibodies to the causative organism. Sometimes, anti-b. burgdorferi antibodies may be detected in the CSF only (2, 5, 23), and they may in rare instances belong to the IgM isotype but not to the IgG isotype (2). Since anti-b. burgdorferi antibodies can be detected in serum from healthy subjects in areas where Lyme disease is endemic, examination of CSF seems to be crucial for a correct diagnosis (1, 3, 34). However, Lyme neuroborreliosis has also been diagnosed in patients with neurologic or psychiatric signs and symptoms and who have shown positive serology in serum for B. burgdorferi, normal CSF findings, and beneficial effects of penicillin treatment (11, 27, 33). The evaluation of additional procedures in the diagnosis and follow-up of patients with neuroborreliosis is therefore warranted. We recently adapted a nitrocellulose immunospot assay to detect individual cells secreting B. burgdorferi antibodies, and we reported accumulation of such cells in the CSF from patients with neuroborreliosis (2). In this report, we describe the kinetics of anti-b. burgdorferi IgG, IgA, and IgM, * Corresponding author. t Present address: Department of Neurology, Huddinge University Hospital, S Huddinge, Sweden antibody-secreting cells in CSF and blood from patients with neuroborreliosis in relation to routine CSF studies, serological findings, and clinical long-term follow-up. An intrathecal immune response, which involves the secretion of anti-b. burgdorferi antibodies of all three isotypes, seems to be characteristic of neuroborreliosis. This local antibody response declines in parallel with clinical improvement and normalization of CSF, but anti-b. burgdorferi IgG antibodysecreting cells may persist at low numbers in CSF for more than 6 months in patients with treated neuroborreliosis. MATERIALS AND METHODS Patients. Paired CSF and blood specimens were obtained from 15 consecutive patients (10 females) with clinical (Table 1), CSF, and serological data speaking in favor of Lyme neuroborreliosis. Patients 1 through 11 and 14 had radiculitis, lesions of one or more cranial nerves, and/or acute meningitis. Patients 12 and 13 had spastic paraparesis, whereas patient 15 is considered to have residual symptoms from myelopathy treated with high-dose penicillin on an empirical basis 5 years before the present study. This patient is included because of the long follow-up. Mononuclear pleocytosis was present in all 14 patients for whom the CSF was examined before treatment, and all 15 patients had oligoclonal IgG bands in the CSF. Routine serology for B. burgdorferi antibodies was positive in the first plasma and CSF specimens available from 12 of the patients, whereas the 3 remaining patients had positive serology in CSF only. Thirteen of the patients were examined before and after treatment, whereas the remaining two patients (patients 1 and 15) were examined only after therapy. From all 15 patients, two or more pairs of CSF and peripheral blood samples were obtained. Specimens I through IV were taken before initiation of antibiotic treatment, within 1 month after treatment, 1 to 6 months after treatment, and more than 6 months after treatment, respectively. In several instances, more than one pair of specimens was available within each of these time periods, and in these cases we selected the last available pair of specimens for this study. The severity of

2 VOL. 59, 1991 ANTI-B. BURGDORFERI ANTIBODIES AND LYME DISEASE 1051 TABLE 1. Clinical data for 15 patients with neuroborreliosisa Time after Patient Age Neurological onset of Treatno. (yr) manifestationsb neurological ment symptoms 1 77 N VII palsy, vertigo, gait distur- 5 days + bances 2 35 Acute aseptic meningitis 7 days Radicular pains, N V and VII 12 days - lesions 4 44 N V and VII lesions, diplopia 14 days Meningoradiculitis, N VII palsy 17 days Meningoradiculitis, N VII palsy 21 days Meningoradiculitis, neurasthenia 21 days Meningoradiculitis 28 days Aseptic meningitis 30 days Meningoradiculitis, bilateral N 30 days - VII palsy Meningoradiculitis 72 days Spastic paraparesis 94 days Spastic paraparesis 6 months N V lesion 11 months Myelopathy 5 years + a Time after onset refers to days after the occasion when CSF and plasma specimens I were sampled. b N V, Trigeminal nerve; N VII, facial nerve. signs and symptoms, as related to damage of the nervous system, was graded as severe (+ + +), moderate (+ +), or minor (+). For determination of anti-b. burgdorferi antibody-secreting cells, CSF and blood were also obtained from 15 patients with other inflammatory neurological diseases. From each of these patients, CSF and blood were obtained on two or more occasions over the course of disease. The patients age varied between 25 and 76 years (mean, 47 years). Ten of these patients had aseptic meningitis (eight acute, two chronic) of unknown etiology, two had varicella zoster virus myelitis, one patient had herpes simplex virus type 1 meningitis, and two had tertiary neurosyphilis. Routine CSF studies revealed >5 x 106 mononuclear cells per liter in 14 of these patients on the first examination (range, 3 x 106 to 1,600 x 106 cells per liter; mean, 368 x 106 cells per liter), oligoclonal IgG bands in CSF in 9 patients, and IgG bands in both CSF and plasma in one patient. Another group of 13 patients with noninflammatory neurological diseases was also evaluated for anti-b. burgdorferi IgG antibody-secreting cells in CSF and blood. Six of these patients had muscular tension headache, three had cerebrovascular disease, two had noninflammatory polyneuropathy, and one each had dizziness and paresthesia. Their age varied between 21 and 88 years (mean, 42 years). Routine CSF studies. CSF cells were counted by phasecontrast microscopy within 30 min after lumbar puncture. Contamination with erythrocytes was rare and did not exceed 200 x 106 cells per liter. Albumin and IgG were measured in CSF and corresponding plasma by immunoprecipitation nephelometry. The blood-brain barrier was evaluated by determination of the CSF/plasma albumin ratio. Age-related upper reference values were used (36). The IgG index (ratio of the CSF/serum IgG ratio to the CSF/serum albumin ratio) was determined (21); values of >0.7 are considered to reflect intrathecal IgG synthesis. To detect oligoclonal IgG bands, we performed agarose isoelectric focusing on unconcentrated CSF and diluted plasma followed by protein transfer to a nitrocellulose membrane, immunolabelling, avidin-biotin amplification, and peroxidase staining (26). Two or more bands present in CSF only are considered to reflect intrathecal IgG synthesis. Preparation of B. burgdorferi antigen for immunospot assay. B. burgdorferi Aca-1 was isolated from the skin of a patient with acrodermatitis chronica atrophicans, prepared as described previously (14), and stored at -20 C in suitable batches until use. CSF and blood sampling. Between 15 and 20 ml of CSF was sampled in siliconized glass tubes. After cell counting, the CSF was centrifuged at 1,000 rpm for 10 min. The supernatant was discarded, and cell pellet was suspended in tissue culture medium (TCM) consisting of the Iscove modification of Dulbecco medium (Flow, Irvine, United Kingdom), 2 mmol of L-glutamine (Flow), 5% (vol/vol) fetal calf serum (Gibco, Paisley, United Kingdom), and antibiotics. This suspension was centrifuged, washed twice in TCM, and diluted to a final cell concentration of 4 x 104 to 50 x 104 cells per ml. Peripheral blood was taken simultaneously in heparinized glass tubes, and cells were separated by density gradient centrifugation on Ficoll (Lymphoprep, Nyegaard, Oslo, Norway), washed three times in phosphate-buffered saline (ph 7.4) (PBS), diluted in TCM, counted, and adjusted to a final concentration of 106 cells per ml. Nitrocellulose immunospot assay. To detect anti-b. burgdorferi antibody-secreting cells, a solid-phase enzyme-linked immunospot assay (6, 32) with microtiter plates with 96 wells and nitrocellulose bottoms (Millititre-HA; Millipore, Bedford, Mass.) was used as previously described (2). Coating of nitrocellulose membranes was performed overnight at 4 C with 100 pal of a solution containing 1 pug of the B. burgdorferi antigen preparation per ml in each well. The optimal antigen concentration for coating has previously been established. After the coating solution was removed through the nitrocellulose membrane by suction into a Millititre vacuum filtration holder (Millipore), followed by blocking with 2% bovine serum albumin in PBS and then washing in PBS, 100-pu aliquots containing 4 x 103 to 50 x 103 CSF cells or 105 peripheral blood lymphocytes (PBL) were applied into individual wells. After incubation overnight at 37 C in a 7% CO2 humidified atmosphere, wells were emptied and washed, and 100 pa of diluted high-affinity purified biotinylated goat anti-human IgG, IgA, or IgM antiserum (Sigma, St. Louis, Mo.) was added to appropriate wells. After 3 h, the wells were washed with PBS, incubated for 1 h with diluted avidin-biotin peroxidase complex (ABC Vectastain Kit; Vector, Burlingame, Calif.), washed, stained with substrate (17), washed, and dried. Formed immunospots were counted in a dissection microscope at a magnification of x25. Values obtained were standardized to the numbers of spots per 104 CSF cells or PBL. For CSF, values are also given as numbers of specific antibody-secreting cells per milliliter of CSF, based on the results from routine CSF cell count (see above). We have previously presented evidence that the spots that we detect in this assay represent secretion of antibodies (38). Determination of B. burgdorfefi flagellum IgG antibodies by ELISA. Anti-B. burgdorferi flagellum IgG antibodies were measured in CSF and plasma by an indirect enzyme-linked immunosorbent assay (ELISA) (14). Microtiter ELISA plates were coated overnight with purified B. burgdorferi flagellum as the antigen, washed, and blocked by bovine serum albumin in PBS-Tween 20. Corresponding CSF and plasma samples were adjusted to 5 mg of IgG per liter, and 100 p.l was added to each well. Plates were incubated for 2 h at 20 C. After the wells were washed with PBS-Tween 20,

3 1052 BAIG ET AL. INFECT. IMMUN. I 11 III IV. SPECIMENS FIG. 1. Mean values for numbers of mononuclear cells in CSF x 106 per liter (U), CSF/plasma albumin ratio (A), CSF IgG index (0), and severity of signs and symptoms (O) (compare patients) over the course of neuroborreliosis. The upper reference limits are 5 x 106 cells per liter for mononuclear cells in CSF and 0.7 for the IgG index, while those for the CSF/plasma albumin ratio are age related (36). Specimens were obtained as follows: I, before initiation of penicillin treatment; II, within 1 month after treatment; III, 1 to 6 months after treatment; IV, more than 6 months after treatment. they were incubated for 2 h with peroxidase-conjugated rabbit anti-human IgG followed by a 15-min substrate reaction. Optical density was read by automated spectrophotometry at 492 nm. Intrathecal anti-b. burgdorferi flagellum IgG antibody production was considered to occur when the calculated CSF/plasma optical density ratio was >1. Statistical analysis. The Mann-Whitney U test was used for statistical analysis. RESULTS CSF abnormalities in neuroborreliosis. (i) Mononuclear pleocytosis. All patients had mononuclear pleocytosis in CSF specimens obtained before treatment (Fig. 1). When examined within 1 month after treatment, 9 of 10 patients had TABLE 2. pleocytosis. This was also detected in 5 of 11 patients examined 1 to 6 months after treatment, whereas normal CSF cell counts were encountered in all 10 patients examined later. (ii) Blood-brain barrier damage. Elevated CSF/plasma albumin ratios were present in 10 of 14 patients examined before treatment (Fig. 1). When examined within 1 month after treatment, 3 of 10 patients had still blood-brain barrier damage; this was also present in 3 of 11 patients examined 1 to 6 months after treatment and in 1 of 9 patients examined more than 6 months after treatment. (iii) IgG index. Eight of 14 patients examined before treatment had elevated IgG indexes (Fig. 1). Within 1 month after treatment, 5 out of 10 patients had elevated indexes, as had 2 of 11 patients examined 1 to 6 months after treatment and 2 of 10 patients examined later than 6 months. Oligoclonal IgG bands. All 14 patients examined before treatment had oligoclonal IgG bands; 11 patients had bands only in CSF, and 3 had bands in the CSF and plasma concomitantly. Within 1 month after treatment, 9 of 10 patients had oligoclonal IgG bands in CSF; 1 to 6 months after treatment, 10 of 11 patients had oligoclonal IgG bands, 9 of them in CSF only. In the last specimens taken more than 6 months after treatment, 6 of the 10 patients examined had oligoclonal IgG bands, 5 of them in CSF only. Disappearance of oligoclonal IgG bands in CSF could be documented in four of the patients over the observation period. Among the three patients with oligoclonal bands in CSF and plasma in parallel, the bands persisted in one patient over the observation period, disappeared in one, and changed to bands in CSF only in one. Anti-B. burgdorferi IgG antibody-secreting cells. All 13 patients examined before initiation of antibiotic treatment had anti-b. burgdorferi IgG antibody-secreting cells in their CSF, ranging between 2 and 115 per 104 CSF cells (mean, 32 cells per 104 CSF cells), corresponding to between 28 and 1,909 cells per ml of CSF (mean, 487 cells per ml) (Tables 2, 3, and 4; Fig. 2). In peripheral blood, only four of the patients (patients 3, 7, 10, and 13) had specific IgG antibody- Numbers of anti-b. burgdorferi IgG, IgA, and IgM antibody-secreting cells per ml of CSF over the course of neuroborreliosisa No. of antibody-secreting cells Patient no. IgG IgA IgM I II III IV I II III IV I II III IV 1 NDb ND ND ND ND ND ND ND ND ND 0 ND ND ND ND 40 0 ND ND ND ND ND ND ND ND ND ND 0 ND ND ND ND ND 20 ND ND ND ND ND ND 45 ND ND ND ND 23 8 ND ND ND ND ND ND 28 8 ND ND ND ND ND 55 3 ND , ND ND 1, ND ND ND 0 ND 9 ND , ND ND ND ND ND 0 7 ND ND 0 ND 0 ND ND ND ND 0 0 ND ND 15 ND ND ND 9 ND ND ND 0 ND ND ND ND Specimens were taken at the following times: I, before treatment; II, within 1 month after treatment; III, 1 to 6 months after treatment; IV, later than 6 months after treatment. b ND, Not determined.

4 VOL. 59, 1991 ANTI-B. BURGDORFERI ANTIBODIES AND LYME DISEASE 1053 TABLE 3. Patient no. Clinical signs and symptoms (A), numbers of anti-b. burgdorferi IgG antibody-secreting cells per ml of CSF (B), and ELISA CSF/plasma anti-b. burgdorferi IgG optical density ratio (C) Specimen I Specimen II Specimen III Specimen IV Aa B C A B C A B C A B C NDb ND ND ND ND ND (+) 0 ND ND 0 ND ND ND ND ND ND ND ND + 5 ND 0 0 ND ND ND 0 ND ND (+) (+) (+) 0.2 ND ND ND (+) 0.2 ND , (+) ND ND ND ND ND (+) ND ,909 ND ND ND (+) 7 ND ND ND ND ND ND (+) ND ND + ND ND + ND ND a See the text for ratings of clinical signs and symptoms. b ND, Not determined. secreting cells amounting to 0.6, 0.2, 0.2, and 0.4 per per 104 CSF cells (mean, 6 cells per 104 CSF cells in the PBL, respectively. 10 patients), corresponding to between 0.2 and 9 cells per ml In specimens II, which were taken within 1 month after of CSF (mean, 1 cell per ml). Two of the patients (patients 1 treatment, all 10 patients examined still had such cells in and 14) simultaneously had antibody-secreting cells in blood their CSF, ranging between 3 and 179 per 104 CSF cells amounting to 0.1 and 0.6, respectively, per 104 PBL. (mean, 32 cells per 104 CSF cells), corresponding to between The numbers of anti-b. burgdorferi IgG antibody-secret- 4 and 564 cells per ml of CSF (mean, 113 cells per ml). In ing cells per ml CSF were lower in specimens II (P < 0.05), eight of these patients, the antibody-secreting cells were III (P < 0.001), and IV (P < ) as compared with those lower in number compared with those in specimens I, in specimens I. whereas the two remaining patients (patients 7 and 13) had Anti-B. burgdorferi IgA antibody-secreting cells. In the CSF higher numbers. In peripheral blood, only one of these obtained before treatment, cells secreting anti-b. burgdorferi patients (patient 3) had antibody secreting cells (0.4 cells per antibodies of the IgA isotype were detected in 6 of PBL). patients. The numbers varied between 2 and 19 per 104 CSF In specimens III, obtained 1 to 6 months after treatment, cells (mean, 5 cells per 104 CSF cells in the 12 patients), 9 of 11 patients examined had anti-b. burgdorferi IgG corresponding to between 23 and 423 cells per ml of CSF antibody-secreting cells in CSF at numbers between 4 and 22 (mean, 81 cells per ml). In peripheral blood, only one patient per 104 CSF cells (mean, 11 cells per 104 CSF cells in the 11 (patient 2) had IgA antibody-secreting cells numbering 3 patients), corresponding to between 1 and 15 cells per ml of cells per 104 PBL. CSF (mean, 6 cells per ml). Numbers in all positive patients Within 1 month after treatment, all of the 8 patients were lower compared with those in previous samples. In examined had IgA antibody-secreting cells in their CSF, peripheral blood, patient 3 was again positive for antibody- ranging between 1 and 26 per 104 CSF cells (mean, 6 cells per secreting cells. 104 CSF cells), corresponding to between 1 and 257 cells per In specimens IV, taken more than 6 months after antibi- ml of CSF (mean, 44 cells per ml). In comparison with otic treatment, 5 of the 10 patients examined had still IgG specimen I, four patients had declining numbers, but four antibody-secreting cells in their CSF, ranging between 2 and had increasing numbers; the difference between CSF speci- TABLE 4. Anti-B. burgdorferi IgG, IgA, and IgM antibody-secreting cells over the course of Lyme neuroborreliosis No. of antibody-secreting cells per 104 mononuclear cells (A) or per ml of CSF (B) Speci- IgGb IgAC IgMd mena Range Mean (SD) Median Range Mean (SD) Median Range Mean (SD) Median A B A B A B A B A B A B A B A B A B I , (30) 483 (574) (7) 77 (133) ,170 6 (13) 130 (317) 2 26 II (54) 113 (185) (8) 41 (88) (3) 11 (20) III (8) 7(5) IV (10) 1(1) See footnote a to Table 2. a b For IgG, the numbers of positive samples/total were as follows: I, 13/13; II, 10/10; III, 9/11; IV, 5/10. c For IgA, the numbers of positive samples/total were as follows: I, 6/12; II, 8/8; III, 0/2; IV, 0/1. d For IgM, the numbers of positive samples/total were as follows: I, 10/13; II, 4/8; III, 0/6; IV, 0/3.

5 1054 BAIG ET AL. 0 E UL- C/) C-) ci) 0- U) 500 T ± 0 A A 0X X 0-0 IgG *0* IgA A-A IgM 1I III specimens FIG. 2. Numbers of anti-b. burgdorferi IgG, IgA, and IgM antibody-secreting cells per ml of CSF, related to routine CSF cell count, from patients with neuroborreliosis evaluated over the course of disease. For a definition of specimen numbers, see the legend to Fig. 1. mens I and II was not significant. In peripheral blood, one of 10 patients had IgA antibody-secreting cells in specimen II (0.4 per 104 PBL). Specimens III from two patients and specimen IV from one patient were examined, with negative results. Peripheral blood specimens III from four patients and specimens IV from eight patients were evaluated, also with negative results. Anti-B. burgdorferi IgM antibody-secreting cells. Ten of 13 patients had anti-b. burgdorferi IgM antibody-secreting cells in CSF specimens I, with numbers ranging between 1 and 47 per 104 CSF cells (mean, 6 cells per 104 CSF cells in 13 patients), corresponding to between 9 and 1,170 cells per ml of CSF (mean, 126 cells per ml). In peripheral blood, 2 of 13 patients (patients 2 and 10) had IgM antibody-secreting cells (6 and 0.4 per 104 PBL, respectively). The numbers of anti-b. burgdorferi IgM antibody-secreting cells per ml of CSF were lower in specimens II, III, and IV compared with those in specimens I (P < 0.01, P < 0.001, and P < 0.001, respectively). Four of eight patients had IgM antibody-secreting cells in CSF specimens II, ranging between 1 and 7 per 104 CSF cells (mean, 2 cells per 104 CSF cells in eight patients), corresponding to between 3 and 59 cells per ml of CSF (mean, 15 cells per ml). One of the nine patients (patient 2) examined on this occasion had such cells in peripheral blood (0.2 per 104 PBL). No IgM antibodysecreting cells were detected in the six CSF specimens III or in the four CSF specimens IV that were examined. Nor were IgM antibody-secreting cells detected in blood from 11 and 10 patients, respectively, examined again within the corresponding time periods. Anti-B. burgdorferi antibody-secreting cells in controls. Both neurosyphilis patients had anti-b. burgdorferi IgG antibody-secreting cells in CSF at numbers of 12 and 51 per 104 CSF cells at the first examination and 18 and 37 per 104 CSF cells, respectively, at the last evaluation. The corresponding numbers per milliliter CSF in these two patients were 106 and 32 cells at the first examination and 5 and 7 cells, respectively, at the last occasion. In addition, the two patients with chronic aseptic meningitis also displayed an- IV INFECT. IMMUN. ti-b. burgdorferi IgG antibody-secreting cells in CSF at one occasion (2 and 5 per 104 CSF cells), corresponding to 20 and 16 cells per ml of CSF. All other patients with other inflammatory or noninflammatory neurological diseases were negative in CSF and blood. Anti-B. burgdorferi IgG antibodies determined by ELISA. Intrathecal anti-b. burgdorferi flagellum IgG antibody production, as reflected by a CSF/plasma antibody optical density ratio of >1, was found in all patients examined before treatment, in 8 of 10 patients examined within 1 month after treatment, in 5 of 7 patients examined 1 to 6 months after treatment, and in 3 of 4 patients evaluated later than 6 months after treatment. When comparing numbers of antibody-secreting cells in CSF with CSF/plasma antibody ratios, good agreement was found among the pretreatment specimens and also for most specimens examined later. Anti-B. burgdorferi antibody-secreting cells related to clinical signs and serological and routine CSF findings. A positive correlation was obvious between clinical improvement and decrease of anti-b. burgdorferi antibody-secreting cells (Table 3). Discrepancies occurred in patient 7 (absence of clinical signs and symptoms but presence of cells in CSF secreting IgG antibodies) and in patient 4 (no antibodysecreting cells but still moderate clinical signs and symptoms). The five patients with anti-b. burgdorferi IgG antibody-secreting cells detectable in CSF more than 6 months after treatment had minor residual clinical symptoms, which could be related to neuroborreliosis. Four of them had also persistent oligoclonal IgG bands in CSF. In contrast, among the five patients who had become negative for antibodysecreting cells, only one had minor residual symptoms. Only one of them had persistent oligoclonal IgG bands in CSF. DISCUSSION A specific antibody response as reflected by occurrence of cells secreting anti-b. burgdorferi antibodies was regularly found in pretreatment CSF specimens from patients with neuroborreliosis. The anti-b. burgdorferi antibody response demonstrable at the cell level is strongly compartmentalized to the CSF, since it is infrequently detected in blood and then at low levels. Penicillin treatment was regularly accompanied by rapid clinical improvement and by a simultaneous, rapid decline in numbers of antibody-secreting cells. However, such cells were still detected in half of the patients who were examined later than 6 months after treatment, although the numbers of cells had decreased. Cells secreting anti-b. burgdorferi antibodies of IgA and IgM isotypes were also frequently detected in CSF, but always concomitantly with cells secreting antibodies of the IgG isotype. IgA and IgM antibody-secreting cells were not demonstrable more than 1 month after treatment, but only few specimens were analyzed. All 14 patients with clinical evidence of active Lyme neuroborreliosis had mononuclear pleocytosis in the first CSF specimen examined, and all showed a decrease of the CSF cell count over the observation time. However, when examined 1 to 6 months after penicillin treatment, 5 of 11 patients examined had still a pleocytosis, whereas none of 10 examined later than 6 months had this CSF abnormality. Whether this sequence of events is a consequence of antibiotic treatment or reflects a self-limiting phenomenon is hard to conclude, since a normalization of the CSF cell count without treatment has previously been reported (15). Most of our patients had elevated CSF/plasma albumin ratios, but we were unable to detect any correlation between

6 VOL. 59, 1991 ANTI-B. BURGDORFERI ANTIBODIES AND LYME DISEASE 1055 duration of symptoms and severity of blood-brain barrier damage. The frequency of elevated IgG index and oligoclonal bands in our patients was similar to that previously reported (2, 15, 16, 31). We have not observed patients who had Lyme neuroborreliosis in the absence of CSF abnormalities. In the majority of the patients, the oligoclonal bands were found only in CSF as a reflection of intrathecal IgG production. However, two patients had bands of identical numbers and migration properties in CSF and corresponding plasma samples. The origin of these bands can only be speculated upon. Their presence in CSF can be a consequence of transudation from serum. Alternatively, the same B-cell clones may be present intrathecally and systemically, as previously suggested in connection with human T-cell leukemia virus type I-associated chronic progressive myelopathy (20). Anti-B. burgdorferi IgG antibodies migrating as oligoclonal bands have been reported in CSF from patients with neuroborreliosis, but these specific antibody bands have been shown to comigrate with visible oligoclonal IgG bands only to a minor extent (13). This means that the major proportion of the oligoclonal IgG bands detectable in CSF from patients with neuroborreliosis consists of antibodies directed to other, unknown antigens. Therefore, a complete correlation between the presence of oligoclonal IgG bands in CSF and of cells secreting anti-b. burgdorferi IgG antibodies in CSF cannot be expected. The presence of anti-b. burgdorferi antibody-secreting cells in the CSF of patients with neurosyphilis is an expected finding because of sharing of epitopes between B. burgdorferi and Treponema pallidum (14, 22). The 60-kDa protein, which is present in the outer membrane of the spirochaete, reacts with similar proteins from many different bacteria (12). The presence of anti-b. burgdorferi antibody-secreting cells in two patients with chronic aseptic meningitis could also reflect such cross-reactivity. B-cell response is considered to be short lived in the absence of antigen (10). There is no direct proof of the presence of B. burgdorferi in the CSF in chronic cases. Since the central nervous system is a relatively privileged site, it may be that bacteria hide at certain places intracellularly, as suggested by Steere (33). The antigen variation of B. burgdorferi, like that employed by other members of the genus Borrelia, could be the other reason for prolonged B-cell response after treatment (27), or the mere presence of disintegrated bacterial proteins could carry on this prolonged response. The other disease that has many immunological resemblances to neuroborreliosis is neurosyphilis. In neurosyphilis, about 30% of patients are known to have persistent positive serology for many years even after treatment, so-called asymptomatic neurosyphilis (8, 37). Whether a correspondent situation exists in neuroborreliosis remains to be evaluated on larger groups of patients. In conclusion, we have documented that neuroborreliosis is accompanied by a specific B-cell response that is strongly compartmentalized to the CSF and that may persist at a low level for more than 6 months after therapy, as evaluated by enumeration of antibody-secreting cells. These cells secrete antibodies preferentially of the IgG isotype. Their presence correlates with the clinical signs of neuroborreliosis. REFERENCES 1. Ackermann, R., B. Rehse-Kupper, E. Golimer, and R. Schmidt Chronic neurologic manifestations of erythema migrans borreliosis. Ann. N.Y. Acad. Sci. 539: Baig, S., T. Olsson, and H. Link Predominance of Borrelia burgdorferi specific B cells in cerebrospinal fluid in neuroborreliosis. 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