INFECTION AND IMMUNITY, Mar. 1991, p. 1050-10560019-9567/91/031050-07$02.00/0Copyright ©) 1991, American Society for Microbiology

Anti-Borrelia burgdorferi Antibody Response over the Courseof Lyme Neuroborreliosis

SHAHID BAIG,' TOMAS OLSSON,' KLAUS HANSEN,2 AND HANS LINKlt*

Department of Neurology, Karolinska Institutet, Huddinge University Hospital, Stockholm, Sweden,1 andBorrelia 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 includemononuclear pleocytosis, oligoclonal immunoglobulin G (IgG) bands, and evidence for local production ofspecific antibodies. We utilized an immunospot assay to detect cells secreting anti-Borrelia burgdorfjeriantibodies of different isotypes over the course of disease. Such cells were detected in CSF from 13 consecutivepatients with neuroborreliosis examined before treatment. IgG antibody-secreting cells were present in highnumbers (mean, 32 cells per 104 CSF cells), whereas IgA and IgM antibody-secreting cells were found lessfrequently and at lower numbers (mean, 5 and 6 cells per 104 CSF cells, respectively). Clinical improvementafter penicillin treatment was paralleled by a rapid decline of antibody-secreting cells in CSF, but they were stilldetected, although at lower numbers, in 5 of 10 patients examined more than 6 months after treatment. Thisspecific B-cell response persisted despite clinical improvement. Whether it reflects persistence of antigen isunsettled.

Neurological signs and symptoms resulting from infectionwith the tick-borne spirochete Borrelia burgdorferi may vary

from involvement of a single cranial nerve to severe damageto the central nervous system, such as spastic paraparesis (4,7, 9, 27, 33, 35). Routine examination of the cerebrospinalfluid (CSF) in Lyme neuroborreliosis may show a mononu-

clear 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 thedisease, but it may be absent in stage 3. The CSF/serumalbumin ratio or total CSF protein is frequently increased,reflecting blood-brain barrier damage. Most patients havelocal immunoglobulin production as manifested by an ele-vated immunoglobulin G (IgG) index and oligoclonal IgGbands in CSF (15, 19, 25, 30). Intrathecal synthesis of IgAand/or IgM has also been reported (15, 18, 29). Lymeneuroborreliosis is accompanied by elevated titers of anti-bodies to the causative organism. Sometimes, anti-B. burg-dorferi antibodies may be detected in the CSF only (2, 5, 23),and they may in rare instances belong to the IgM isotype butnot to the IgG isotype (2). Since anti-B. burgdorferi antibod-ies can be detected in serum from healthy subjects in areaswhere Lyme disease is endemic, examination of CSF seemsto be crucial for a correct diagnosis (1, 3, 34). However,Lyme neuroborreliosis has also been diagnosed in patientswith neurologic or psychiatric signs and symptoms and whohave shown positive serology in serum for B. burgdorferi,normal CSF findings, and beneficial effects of penicillintreatment (11, 27, 33). The evaluation of additional proce-dures in the diagnosis and follow-up of patients with neu-roborreliosis 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 frompatients with neuroborreliosis (2). In this report, we describethe kinetics of anti-B. burgdorferi IgG, IgA, and IgM,

* Corresponding author.t Present address: Department of Neurology, Huddinge Univer-

sity Hospital, S-141 86 Huddinge, Sweden.

antibody-secreting cells in CSF and blood from patients withneuroborreliosis in relation to routine CSF studies, serolog-ical findings, and clinical long-term follow-up. An intrathecalimmune response, which involves the secretion of anti-B.burgdorferi antibodies of all three isotypes, seems to becharacteristic of neuroborreliosis. This local antibody re-sponse declines in parallel with clinical improvement andnormalization of CSF, but anti-B. burgdorferi IgG antibody-secreting cells may persist at low numbers in CSF for morethan 6 months in patients with treated neuroborreliosis.

MATERIALS AND METHODS

Patients. Paired CSF and blood specimens were obtainedfrom 15 consecutive patients (10 females) with clinical (Table1), CSF, and serological data speaking in favor of Lymeneuroborreliosis. Patients 1 through 11 and 14 had radiculi-tis, lesions of one or more cranial nerves, and/or acutemeningitis. Patients 12 and 13 had spastic paraparesis,whereas patient 15 is considered to have residual symptomsfrom myelopathy treated with high-dose penicillin on anempirical basis 5 years before the present study. This patientis included because of the long follow-up. Mononuclearpleocytosis was present in all 14 patients for whom the CSFwas examined before treatment, and all 15 patients hadoligoclonal IgG bands in the CSF. Routine serology for B.burgdorferi antibodies was positive in the first plasma andCSF specimens available from 12 of the patients, whereasthe 3 remaining patients had positive serology in CSF only.Thirteen of the patients were examined before and aftertreatment, whereas the remaining two patients (patients 1and 15) were examined only after therapy. From all 15patients, two or more pairs of CSF and peripheral bloodsamples were obtained. Specimens I through IV were takenbefore initiation of antibiotic treatment, within 1 month aftertreatment, 1 to 6 months after treatment, and more than 6months after treatment, respectively. In several instances,more than one pair of specimens was available within each ofthese time periods, and in these cases we selected the lastavailable pair of specimens for this study. The severity of

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TABLE 1. Clinical data for 15 patients with neuroborreliosisa

Time afterPatient Age Neurological onset of Treat-no. (yr) manifestationsb neurological ment

symptoms

1 77 N VII palsy, vertigo, gait distur- 5 days +bances

2 35 Acute aseptic meningitis 7 days -

3 64 Radicular pains, N V and VII 12 days -

lesions4 44 N V and VII lesions, diplopia 14 days -

5 35 Meningoradiculitis, N VII palsy 17 days -

6 68 Meningoradiculitis, N VII palsy 21 days -

7 42 Meningoradiculitis, neurasthenia 21 days -

8 23 Meningoradiculitis 28 days -

9 65 Aseptic meningitis 30 days -

10 25 Meningoradiculitis, bilateral N 30 days -

VII palsy11 65 Meningoradiculitis 72 days -

12 64 Spastic paraparesis 94 days -

13 75 Spastic paraparesis 6 months -

14 73 N V lesion 11 months -

15 55 Myelopathy 5 years +

a Time after onset refers to days after the occasion when CSF and plasmaspecimens I were sampled.

b N V, Trigeminal nerve; N VII, facial nerve.

signs and symptoms, as related to damage of the nervoussystem, was graded as severe (+ + +), moderate (+ +), orminor (+).For determination of anti-B. burgdorferi antibody-secret-

ing cells, CSF and blood were also obtained from 15 patientswith other inflammatory neurological diseases. From each ofthese patients, CSF and blood were obtained on two or moreoccasions over the course of disease. The patients age variedbetween 25 and 76 years (mean, 47 years). Ten of thesepatients had aseptic meningitis (eight acute, two chronic) ofunknown etiology, two had varicella zoster virus myelitis,one patient had herpes simplex virus type 1 meningitis, andtwo had tertiary neurosyphilis. Routine CSF studies re-vealed >5 x 106 mononuclear cells per liter in 14 of thesepatients on the first examination (range, 3 x 106 to 1,600 x106 cells per liter; mean, 368 x 106 cells per liter), oligoclonalIgG bands in CSF in 9 patients, and IgG bands in both CSFand plasma in one patient.Another group of 13 patients with noninflammatory neu-

rological diseases was also evaluated for anti-B. burgdorferiIgG antibody-secreting cells in CSF and blood. Six of thesepatients had muscular tension headache, three had cere-brovascular disease, two had noninflammatory polyneurop-athy, and one each had dizziness and paresthesia. Their agevaried between 21 and 88 years (mean, 42 years).

Routine CSF studies. CSF cells were counted by phase-contrast microscopy within 30 min after lumbar puncture.Contamination with erythrocytes was rare and did not ex-ceed 200 x 106 cells per liter. Albumin and IgG weremeasured in CSF and corresponding plasma by immunopre-cipitation nephelometry. The blood-brain barrier was evalu-ated by determination of the CSF/plasma albumin ratio.Age-related upper reference values were used (36). The IgGindex (ratio of the CSF/serum IgG ratio to the CSF/serumalbumin ratio) was determined (21); values of >0.7 areconsidered to reflect intrathecal IgG synthesis. To detectoligoclonal IgG bands, we performed agarose isoelectricfocusing on unconcentrated CSF and diluted plasma fol-lowed by protein transfer to a nitrocellulose membrane,

immunolabelling, avidin-biotin amplification, and peroxidasestaining (26). Two or more bands present in CSF only areconsidered to reflect intrathecal IgG synthesis.

Preparation of B. burgdorferi antigen for immunospot as-say. B. burgdorferi Aca-1 was isolated from the skin of apatient with acrodermatitis chronica atrophicans, preparedas described previously (14), and stored at -20°C in suitablebatches 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. Thesupernatant was discarded, and cell pellet was suspended intissue culture medium (TCM) consisting of the Iscove mod-ification of Dulbecco medium (Flow, Irvine, United King-dom), 2 mmol of L-glutamine (Flow), 5% (vol/vol) fetal calfserum (Gibco, Paisley, United Kingdom), and antibiotics.This suspension was centrifuged, washed twice in TCM, anddiluted to a final cell concentration of 4 x 104 to 50 x 104cells per ml. Peripheral blood was taken simultaneously inheparinized glass tubes, and cells were separated by densitygradient centrifugation on Ficoll (Lymphoprep, Nyegaard,Oslo, Norway), washed three times in phosphate-bufferedsaline (pH 7.4) (PBS), diluted in TCM, counted, and adjustedto a final concentration of 106 cells per ml.

Nitrocellulose immunospot assay. To detect anti-B. burg-dorferi antibody-secreting cells, a solid-phase enzyme-linkedimmunospot assay (6, 32) with microtiter plates with 96 wellsand nitrocellulose bottoms (Millititre-HA; Millipore, Bed-ford, Mass.) was used as previously described (2). Coating ofnitrocellulose membranes was performed overnight at 4°Cwith 100 pAl of a solution containing 1 pug of the B. burgdorferiantigen preparation per ml in each well. The optimal antigenconcentration for coating has previously been established.After the coating solution was removed through the nitro-cellulose membrane by suction into a Millititre vacuumfiltration 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 or105 peripheral blood lymphocytes (PBL) were applied intoindividual wells. After incubation overnight at 37°C in a 7%CO2 humidified atmosphere, wells were emptied andwashed, and 100 pA of diluted high-affinity purified biotiny-lated 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 withdiluted avidin-biotin peroxidase complex (ABC VectastainKit; Vector, Burlingame, Calif.), washed, stained with sub-strate (17), washed, and dried. Formed immunospots werecounted in a dissection microscope at a magnification ofx25. Values obtained were standardized to the numbers ofspots per 104 CSF cells or PBL. For CSF, values are alsogiven as numbers of specific antibody-secreting cells permilliliter of CSF, based on the results from routine CSF cellcount (see above). We have previously presented evidencethat the spots that we detect in this assay represent secretionof antibodies (38).

Determination of B. burgdorfefi flagellum IgG antibodies byELISA. Anti-B. burgdorferi flagellum IgG antibodies weremeasured in CSF and plasma by an indirect enzyme-linkedimmunosorbent assay (ELISA) (14). Microtiter ELISAplates were coated overnight with purified B. burgdorferiflagellum as the antigen, washed, and blocked by bovineserum albumin in PBS-Tween 20. Corresponding CSF andplasma samples were adjusted to 5 mg of IgG per liter, and100 p.l was added to each well. Plates were incubated for 2 hat 20°C. After the wells were washed with PBS-Tween 20,

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I 11 III IV.SPECIMENS

FIG. 1. Mean values for numbers of mononuclear cells in CSF x106 per liter (U), CSF/plasma albumin ratio (A), CSF IgG index (0),and severity of signs and symptoms (O) (compare patients) over thecourse of neuroborreliosis. The upper reference limits are 5 x 106cells per liter for mononuclear cells in CSF and 0.7 for the IgGindex, while those for the CSF/plasma albumin ratio are age related(36). Specimens were obtained as follows: I, before initiation ofpenicillin treatment; II, within 1 month after treatment; III, 1 to 6months after treatment; IV, more than 6 months after treatment.

they were incubated for 2 h with peroxidase-conjugatedrabbit anti-human IgG followed by a 15-min substrate reac-

tion. Optical density was read by automated spectropho-tometry at 492 nm. Intrathecal anti-B. burgdorferi flagellumIgG antibody production was considered to occur when thecalculated CSF/plasma optical density ratio was >1.

Statistical analysis. The Mann-Whitney U test was used forstatistical analysis.

RESULTS

CSF abnormalities in neuroborreliosis. (i) Mononuclearpleocytosis. All patients had mononuclear pleocytosis in CSFspecimens obtained before treatment (Fig. 1). When exam-ined within 1 month after treatment, 9 of 10 patients had

pleocytosis. This was also detected in 5 of 11 patientsexamined 1 to 6 months after treatment, whereas normalCSF cell counts were encountered in all 10 patients exam-

ined later.(ii) Blood-brain barrier damage. Elevated CSF/plasma

albumin ratios were present in 10 of 14 patients examinedbefore treatment (Fig. 1). When examined within 1 monthafter treatment, 3 of 10 patients had still blood-brain barrierdamage; this was also present in 3 of 11 patients examined 1to 6 months after treatment and in 1 of 9 patients examinedmore than 6 months after treatment.

(iii) IgG index. Eight of 14 patients examined beforetreatment had elevated IgG indexes (Fig. 1). Within 1 monthafter treatment, 5 out of 10 patients had elevated indexes, as

had 2 of 11 patients examined 1 to 6 months after treatmentand 2 of 10 patients examined later than 6 months.

Oligoclonal IgG bands. All 14 patients examined beforetreatment had oligoclonal IgG bands; 11 patients had bandsonly in CSF, and 3 had bands in the CSF and plasmaconcomitantly. Within 1 month after treatment, 9 of 10patients had oligoclonal IgG bands in CSF; 1 to 6 monthsafter treatment, 10 of 11 patients had oligoclonal IgG bands,9 of them in CSF only. In the last specimens taken more than6 months after treatment, 6 of the 10 patients examined hadoligoclonal IgG bands, 5 of them in CSF only. Disappear-ance of oligoclonal IgG bands in CSF could be documentedin four of the patients over the observation period. Amongthe three patients with oligoclonal bands in CSF and plasmain parallel, the bands persisted in one patient over theobservation period, disappeared in one, and changed tobands in CSF only in one.

Anti-B. burgdorferi IgG antibody-secreting cells. All 13patients examined before initiation of antibiotic treatmenthad anti-B. burgdorferi IgG antibody-secreting cells in theirCSF, ranging between 2 and 115 per 104 CSF cells (mean, 32cells per 104 CSF cells), corresponding to between 28 and1,909 cells per ml of CSF (mean, 487 cells per ml) (Tables 2,3, and 4; Fig. 2). In peripheral blood, only four of thepatients (patients 3, 7, 10, and 13) had specific IgG antibody-

TABLE 2. Numbers of anti-B. burgdorferi IgG, IgA, and IgM antibody-secreting cells per ml of CSF overthe 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 16 8 3 ND ND ND ND ND ND ND ND2 141 4 ND 0 ND ND ND ND 40 0 ND ND3 315 23 0 ND 35 38 ND ND 70 53 0 ND4 229 ND 0 0 0 ND ND ND 0 ND 0 05 771 ND 12 0 0 ND ND ND 20 ND ND 06 630 ND 5 0 0 ND ND ND 45 ND ND ND7 28 71 3 ND 23 8 ND ND 114 0 ND ND8 221 36 1 0.2 83 16 ND ND 28 8 ND ND9 327 20 ND 0.2 207 3 ND ND 55 3 ND 010 1,394 73 8 ND 423 21 0 ND 1,170 0 0 ND11 43 ND 12 0 0 ND 0 ND 9 ND 0 012 1,909 564 15 ND 199 257 ND ND 83 59 0 ND13 296 322 7 ND 0 7 ND ND 0 ND 0 ND14 32 4 ND 0.4 0 1 ND ND 0 0 ND ND15 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 monthsafter treatment.

b ND, Not determined.

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TABLE 3. Clinical signs and symptoms (A), numbers of anti-B. burgdorferi IgG antibody-secreting cells per ml of CSF (B), and ELISACSF/plasma anti-B. burgdorferi IgG optical density ratio (C)

Specimen I Specimen II Specimen III Specimen IVPatient no.

Aa B C A B C A B C A B C

1 +++ NDb ND ++ 16 0.5 ++ 8 0.6 + 3 ND2 +++ 141 1.6 + 4 1.0 ND ND ND (+) 0 ND3 +++ 315 7.3 + 23 7.6 0 0 ND 0 ND ND4 +++ 229 3.6 ++ ND ND ++ 0 3.4 0 0 4.55 +++ 771 3.2 ++ ND ND + 12 1.0 0 0 1.06 +++ 630 1.2 ++ ND ND + 5 ND 0 0 ND7 +++ 28 ND + 71 1.8 0 3 ND 0 ND ND8 ++ 221 1.3 (+) 36 2.7 (+) 1 5.9 (+) 0.2 ND9 +++ 327 10.8 ++ 20 12.1 + ND ND (+) 0.2 ND10 +++ 1,394 3.0 ++ 73 4.3 + 8 4.4 (+) ND ND11 ++ 43 2.5 ND ND ND (+) 12 2.8 0 0 ND12 +++ 1,909 ND ++ 564 4.0 + 15 6.5 + ND ND13 +++ 296 1.7 ++ 322 1.9 (+) 7 ND ND ND ND14 +++ 32 8.3 ++ 4 6.7 + ND ND (+) 0.4 4.715 + ND ND + ND ND + ND ND + 9 20.2

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 104 30 per 104 CSF cells (mean, 6 cells per 104 CSF cells in thePBL, 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 1treatment, all 10 patients examined still had such cells in and 14) simultaneously had antibody-secreting cells in bloodtheir 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 < 0.0001) as compared with thoselower 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 CSFhigher numbers. In peripheral blood, only one of these obtained before treatment, cells secreting anti-B. burgdorferipatients (patient 3) had antibody secreting cells (0.4 cells per antibodies of the IgA isotype were detected in 6 of 12104 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 CSFantibody-secreting cells in CSF at numbers between 4 and 22 (mean, 81 cells per ml). In peripheral blood, only one patientper 104 CSF cells (mean, 11 cells per 104 CSF cells in the 11 (patient 2) had IgA antibody-secreting cells numbering 3patients), 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 patientswere 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 persecreting 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 withotic treatment, 5 of the 10 patients examined had still IgG specimen I, four patients had declining numbers, but fourantibody-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 IgMdmena 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 2-115 28-1,909 32 (30) 483 (574) 18 296 0-19 0-423 5 (7) 77 (133) 1 8 0-47 0-1,170 6 (13) 130 (317) 2 26II 3-179 4-564 33 (54) 113 (185) 12 29 1-26 1-257 6 (8) 41 (88) 3 10 0-7 0-59 2 (3) 11 (20) 0.5 6III 0-22 0-15 11(8) 7(5) 13 8 0 0 0 0IV 0-30 0-4 6(10) 1(1) 1 0.2 0 0 0 0

a See footnote a to Table 2.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.

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0

EUL-C/)

C-)

ci)

0-

U)

500 T

0-0 IgG400 +*0* IgAA-A IgM

300+

200 +

100±

0

AA

0X X

1I III

specimensIV

FIG. 2. Numbers of anti-B. burgdorferi IgG, IgA, and IgMantibody-secreting cells per ml of CSF, related to routine CSF cellcount, 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 of10 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. Peripheralblood specimens III from four patients and specimens IVfrom eight patients were evaluated, also with negative re-

sults.Anti-B. burgdorferi IgM antibody-secreting cells. Ten of 13

patients had anti-B. burgdorferi IgM antibody-secreting cellsin CSF specimens I, with numbers ranging between 1 and 47per 104 CSF cells (mean, 6 cells per 104 CSF cells in 13patients), corresponding to between 9 and 1,170 cells per mlof CSF (mean, 126 cells per ml). In peripheral blood, 2 of 13patients (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-secret-

ing cells per ml of CSF were lower in specimens II, III, andIV compared with those in specimens I (P < 0.01, P < 0.001,and P < 0.001, respectively). Four of eight patients had IgMantibody-secreting cells in CSF specimens II, ranging be-tween 1 and 7 per 104 CSF cells (mean, 2 cells per 104 CSFcells in eight patients), corresponding to between 3 and 59cells per ml of CSF (mean, 15 cells per ml). One of the ninepatients (patient 2) examined on this occasion had such cellsin peripheral blood (0.2 per 104 PBL). No IgM antibody-secreting 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 and10 patients, respectively, examined again within the corre-

sponding time periods.Anti-B. burgdorferi antibody-secreting cells in controls.

Both neurosyphilis patients had anti-B. burgdorferi IgGantibody-secreting cells in CSF at numbers of 12 and 51 per

104 CSF cells at the first examination and 18 and 37 per 104CSF cells, respectively, at the last evaluation. The corre-

sponding numbers per milliliter CSF in these two patientswere 106 and 32 cells at the first examination and 5 and 7cells, respectively, at the last occasion. In addition, the twopatients with chronic aseptic meningitis also displayed an-

ti-B. burgdorferi IgG antibody-secreting cells in CSF at oneoccasion (2 and 5 per 104 CSF cells), corresponding to 20 and16 cells per ml of CSF. All other patients with otherinflammatory or noninflammatory neurological diseaseswere negative in CSF and blood.

Anti-B. burgdorferi IgG antibodies determined by ELISA.Intrathecal anti-B. burgdorferi flagellum IgG antibody pro-duction, as reflected by a CSF/plasma antibody opticaldensity ratio of >1, was found in all patients examinedbefore treatment, in 8 of 10 patients examined within 1month after treatment, in 5 of 7 patients examined 1 to 6months after treatment, and in 3 of 4 patients evaluated laterthan 6 months after treatment. When comparing numbers ofantibody-secreting cells in CSF with CSF/plasma antibodyratios, good agreement was found among the pretreatmentspecimens and also for most specimens examined later.

Anti-B. burgdorferi antibody-secreting cells related to clini-cal signs and serological and routine CSF findings. A positivecorrelation was obvious between clinical improvement anddecrease of anti-B. burgdorferi antibody-secreting cells (Ta-ble 3). Discrepancies occurred in patient 7 (absence ofclinical signs and symptoms but presence of cells in CSFsecreting IgG antibodies) and in patient 4 (no antibody-secreting cells but still moderate clinical signs and symp-toms). The five patients with anti-B. burgdorferi IgG anti-body-secreting cells detectable in CSF more than 6 monthsafter treatment had minor residual clinical symptoms, whichcould be related to neuroborreliosis. Four of them had alsopersistent oligoclonal IgG bands in CSF. In contrast, amongthe five patients who had become negative for antibody-secreting cells, only one had minor residual symptoms. Onlyone of them had persistent oligoclonal IgG bands in CSF.

DISCUSSION

A specific antibody response as reflected by occurrence ofcells secreting anti-B. burgdorferi antibodies was regularlyfound in pretreatment CSF specimens from patients withneuroborreliosis. The anti-B. burgdorferi antibody responsedemonstrable at the cell level is strongly compartmentalizedto the CSF, since it is infrequently detected in blood andthen at low levels. Penicillin treatment was regularly accom-panied by rapid clinical improvement and by a simultaneous,rapid decline in numbers of antibody-secreting cells. How-ever, such cells were still detected in half of the patients whowere examined later than 6 months after treatment, althoughthe numbers of cells had decreased. Cells secreting anti-B.burgdorferi antibodies of IgA and IgM isotypes were alsofrequently detected in CSF, but always concomitantly withcells secreting antibodies of the IgG isotype. IgA and IgMantibody-secreting cells were not demonstrable more than 1month after treatment, but only few specimens were ana-lyzed.

All 14 patients with clinical evidence of active Lymeneuroborreliosis had mononuclear pleocytosis in the firstCSF specimen examined, and all showed a decrease of theCSF cell count over the observation time. However, whenexamined 1 to 6 months after penicillin treatment, 5 of 11patients examined had still a pleocytosis, whereas none of 10examined later than 6 months had this CSF abnormality.Whether this sequence of events is a consequence of antibi-otic treatment or reflects a self-limiting phenomenon is hardto conclude, since a normalization of the CSF cell countwithout 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

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duration of symptoms and severity of blood-brain barrierdamage.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 Lymeneuroborreliosis in the absence of CSF abnormalities. In themajority of the patients, the oligoclonal bands were foundonly in CSF as a reflection of intrathecal IgG production.However, two patients had bands of identical numbers andmigration properties in CSF and corresponding plasma sam-ples. The origin of these bands can only be speculated upon.Their presence in CSF can be a consequence of transudationfrom serum. Alternatively, the same B-cell clones may bepresent intrathecally and systemically, as previously sug-gested in connection with human T-cell leukemia virus typeI-associated chronic progressive myelopathy (20). Anti-B.burgdorferi IgG antibodies migrating as oligoclonal bandshave been reported in CSF from patients with neuroborreli-osis, but these specific antibody bands have been shown tocomigrate with visible oligoclonal IgG bands only to a minorextent (13). This means that the major proportion of theoligoclonal IgG bands detectable in CSF from patients withneuroborreliosis consists of antibodies directed to other,unknown antigens. Therefore, a complete correlation be-tween the presence of oligoclonal IgG bands in CSF and ofcells secreting anti-B. burgdorferi IgG antibodies in CSFcannot be expected.The presence of anti-B. burgdorferi antibody-secreting

cells in the CSF of patients with neurosyphilis is an expectedfinding because of sharing of epitopes between B. burgdor-feri 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-secretingcells in two patients with chronic aseptic meningitis couldalso reflect such cross-reactivity.

B-cell response is considered to be short lived in theabsence of antigen (10). There is no direct proof of thepresence of B. burgdorferi in the CSF in chronic cases. Sincethe central nervous system is a relatively privileged site, itmay be that bacteria hide at certain places intracellularly, assuggested by Steere (33). The antigen variation of B. burg-dorferi, like that employed by other members of the genusBorrelia, could be the other reason for prolonged B-cellresponse after treatment (27), or the mere presence ofdisintegrated bacterial proteins could carry on this prolongedresponse. The other disease that has many immunologicalresemblances to neuroborreliosis is neurosyphilis. In neuro-syphilis, about 30% of patients are known to have persistentpositive serology for many years even after treatment,so-called asymptomatic neurosyphilis (8, 37). Whether acorrespondent situation exists in neuroborreliosis remains tobe evaluated on larger groups of patients.

In conclusion, we have documented that neuroborreliosisis accompanied by a specific B-cell response that is stronglycompartmentalized to the CSF and that may persist at a lowlevel for more than 6 months after therapy, as evaluated byenumeration of antibody-secreting cells. These cells secreteantibodies preferentially of the IgG isotype. Their presencecorrelates with the clinical signs of neuroborreliosis.

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