PCR detection of Borrelia burgdorferi DNA in cerebrospinal fluid of Lyme neuroborreliosis patients.

We used the polymerase chain reaction (PCR), a method useful in the detection of Borrelia burgdorferi in vitro, to evaluate CSF in patients thought to have neuroborreliosis. Nested pairs of oligonucleotide primers were designed to recognize the C-terminal region of B burgdorferi OspA. CSF samples were obtained from (1) patients with immunologic evidence of systemic B burgdorferi infection and clinical manifestations suggestive of CNS dysfunction, (2) seronegative patients with clinical disorders consistent with Lyme borreliosis, and (3) patient and contamination controls; all were analyzed in a blinded fashion. PCR detected B burgdorferi OspA DNA in CSF of (1) 10 of 11 patients with Lyme encephalopathy, (2) 28 of 37 patients with inflammatory CNS disease, (3) seven of seven seronegative patients with Lyme-compatible disorders, and (4) zero of 23 patient controls. Zero of 83 additional contamination controls were PCR-positive. In eight patients from whom we obtained CSF before and after parenteral antimicrobial therapy, PCR results invariably predicted clinical outcome accurately.     

Virus-specific and autoreactive T cell lines isolated from cerebrospinal fluid of a patient with chronic rubella panencephalitis

Using a recently described technique for expanding of human T lymphocyte populations from cerebrospinal fluid (CSF), we investigated the local cellular immune response in a patient with chronic rubella panencephalitis. A total of 328 T cell lines (TCLs) was established by seeding CSF cells at limiting dilution into histoplates in the presence of irradiated feeder cells and phytohemagglutinin (PHA)-containing conditioned medium. 80% of TCLs expressed the CD4+CD8-, 5% the CD4-CD8+ phenotype and 15% of TCLs contained different proportions of CD4+ and CD8+ cells. Of 191 TCLs analyzed, 85 were cytotoxic, as shown by their lectin-dependent cytotoxicity against allogeneic uninfected target cells. Eight of them demonstrated specificity for the autologous, rubella virus-infected target cells. When tested for antigen-specific proliferative activity, 26 TCLs responded to rubella antigen, 16 TCLs reacted to myelin basic protein (MBP), four TCLs to proteolipid protein (PLP), four to galactocerebrosides and two to actin. Fourteen out of 16 MBP-specific TCLs also responded, to a minor degree, to rubella antigen and/or actin. The results showed that the persisting rubella infection had given rise to autoreactive T cells. Virus-induced autoreactivity to brain antigens may be an important pathogenetic mechanism in other chronic inflammatory disorders of the CNS.     

Latent Lyme neuroborreliosis: presence of Borrelia burgdorferi in the cerebrospinal fluid without concurrent inflammatory signs

Borrelia burgdorferi, the etiologic agent of Lyme borreliosis, was isolated from the CSF of a patient with elevated serum IgG antibody titers against B burgdorferi and a history of multiple tick bites. The absence of concurrent inflammatory signs of CSF as well as intrathecal antibody production indicates a phase of latent Lyme neuroborreliosis in which no tissue infection or reaction has yet occurred. Bilateral tinnitus was the only clinical symptom in this patient. The persistence of the bilateral tinnitus after antibiotic therapy did not support a causal relationship between this symptom and the borrelial infection.     

Lyme neuroborreliosis: a new sensitive diagnostic assay for intrathecal synthesis of Borrelia burgdorferi--specific immunoglobulin G, A, and M

An antibody capture enzyme-linked immunosorbent assay was developed to measure directly intrathecal immunoglobulin (Ig) G, A, and M synthesis specific for Borrelia burgdorferi. Purified, biotin-avidin-peroxidase-labeled B. burgdorferi flagella was used as test antigen. Paired cerebrospinal fluid and serum specimens from 100 patients with clinically definite neuroborreliosis and 35 control subjects with neurological diseases were examined. Significant B. burgdorferi-specific intrathecal IgG, A, and M production was found in 89%, 65% and 67% of patients with neuroborreliosis. Local synthesis of specific IgA was only seen in patients with significant local IgG synthesis. Antibody production in cerebrospinal fluid began by 2 weeks after onset of neurological symptoms. At the end of the second week specific IgM, IgG, or both, was detected in 88% of the patients. Specific IgG synthesis was present in all patients by 6 weeks after onset. Specific local IgM synthesis usually disappeared by 3 to 6 months after therapy, whereas specific IgG synthesis persisted after recovery. Even in patients with a severely altered blood-brain barrier, the assay discriminated between intrathecal antibody synthesis and antibody leakage from serum. The assay makes diagnostic measurement of B. burgdorferi-specific intrathecal antibody synthesis reliable, rapid, and accessible as a routine serological test.     

Virus-reactive and autoreactive T cells are accumulated in cerebrospinal fluid in multiple sclerosis

Elevated numbers of B cells--plasma cells secreting antibodies to measles and mumps virus, and to myelin associated glycoprotein (MAG), one of several putative myelin autoantigens--have previously been reported in cerebrospinal fluid (CSF) from patients with multiple sclerosis (MS), while it is unknown if corresponding T cell reactivities occur. We have defined the T cell reactivities to measles and mumps virus and to MAG in an immunospot assay which is based on the detection of secretion of interferon-gamma (IFN-gamma) by single cells upon stimulation with specific antigen in short term cultures. Patients with MS had higher numbers of MAG-reactive T cells in blood compared to controls, while no differences were observed for measles or mumps virus-reactive T cells. In CSF, elevated numbers of MAG-reactive T cells and also of measles- and mumps-reactive T cells were found in patients with MS compared to other neurological diseases. A strong accumulation of antigen-reactive T cells was observed in the MS patients' CSF compared to blood. The magnitude of these T cell reactivities did not correlate with clinical MS variables. The T cell repertoire in MS thus includes, besides myelin basic protein, proteolipid protein and myelin oligodendrocyte glycoprotein, also MAG and, in addition, measles and mumps virus. It is not clear whether these T cell reactivities accumulated in the CSF have importance for the pathogenesis of MS or reflect phenomena secondary to myelin damage, or result from both these alternatives.     

Astroglial and neuronal proteins in cerebrospinal fluid as markers of CNS involvement in Lyme neuroborreliosis

Is Lyme neuroborreliosis, even in its early phase, a parenchymatous disorder in the central nervous system (CNS), and not merely a meningitic process? We quantified cerebrospinal fluid (CSF) levels of four nerve and glial cell marker proteins in Lyme neuroborreliosis patients with pretreatment durations of 7–240 days. AH 23 patients had meningo-radiculitis, and six had objective signs of encephalopathy. Glial fibrillary acidic protein (GFAp) pretreatment levels in CSF, and the light subunit of neurofilament protein (NFL) levels were related to clinical outcome and declined significantly after treatment (P < 0.001 and P < 0.01, respectively). NFL was detectable in 11 out of 22 patients, and pre-and post-treatment NFL levels were associated with the duration of neurological symptoms within 100 days prior to treatment. Neuron-specific enolase (NSE) concentrations also decreased after therapy (P < 0.001), while CSF levels of glial S-100 protein remained unchanged. The pretreatment duration of disease was related to postinfectious sequelae. GFAp, NSE and NFL levels in CSF are unspecific indicators of astroglial and neuronal involvement in CNS disease. The findings in the present study are in agreement with the hypothesis that early and late stages of Lyme neuroborreliosis damage the CNS parenchyma.     

T-cell subsets in multiple sclerosis: relationships between peripheral blood and cerebrospinal fluid

We contemporarily studied cerebrospinal fluid (CSF) and peripheral blood (PB) T-cell subsets, defined by monoclonal antibodies, in 29 patients with multiple sclerosis (MS) and 10 patients with other neurological diseases (OND). All subjects showed a clear-cut prevalence of CSF T-cells. Similarly, T-helper and T-suppressor subsets tended to show higher percentages in CSF in almost all subjects except relapsing MS, who were characterized by low percentages of T-suppressors in PB and even much lower percentages in CSF. Helper/suppressor ratios were found to be almost similar in the two body compartments of OND patients, lower in CSF than in PB of chronic progressive MS, always higher in CSF than in PB of relapsing MS. MS patients in remission showed both patterns of progressive MS and OND patients. Our results demonstrate that the loss of PB T-suppressor in relapsing MS is not due to a migration of such cells into CSF. Furthermore, regarding T-lymphocyte subsets, a typical CSF/PB pattern characterizes relapsing MS from other patients.     

T-cell subsets in the cerebrospinal fluid and blood of patients with multiple sclerosis

The absolute numbers and ratios of helper/inducer (T4) and cytotoxic/suppressor (T8) T-cells were determined in cerebrospinal fluid (CSF) and blood of patients with multiple sclerosis (MS) and various other neurologic diseases (OND). In patients with MS, the T4:T8 ratio was higher in both blood and CSF, and the increase was significantly greater in CSF than in blood. These findings were due to an increased proportion of T4-lymphocytes in the CSF and to a decreased proportion of T8-cells in blood. These results indicate the need for additional studies of CSF lymphocytes in patients with MS.     

T cell lines established from multiple sclerosis cerebrospinal fluid T cells using human retroviruses

Cerebrospinal fluid (CSF) lymphocytes from patients with multiple sclerosis (MS) were transformed with human T cell leukemia/lymphoma virus (HTLV I and HTLV II) and the resulting cell lines characterized by cell surface phenotyping and functional assessment. The lines were predominantly of the CD4 helper/induce phenotype although the HTLV II lines contained 10-20% CD8+ cells. The lines appeared to be activated cells; the majority were TA1+, HLA-DR+, and TAC+ (CD25+). Interestingly, they were OKT10- (CD38-). Functionally, the lines contained no natural killer (NK) activity and were modestly cytotoxic in the antibody-dependent cellular cytotoxicity (ADCC) assay. They were poor proliferative responders to antigens and mitogens though the HTLV II lines did respond to interleukin 2 (IL2). The HTLV I lines were either nonresponsive to or were suppressed by IL2. Early passages of two of the lines produced IL2 but this was lost as the cells were passed in culture. The cell lines were capable of either directly or indirectly suppressing pokeweed mitogen (PWM)-driven immunoglobulin production by normal B cells. In addition, the lines were capable of producing gamma-interferon (IFN-gamma), lymphotoxin (LT), an interleukin 1 (IL1)-like factor, glial growth promoting factor (GGPF), and IL6. The advantage of these lines over clones or cell lines developed using other techniques is their growth in the absence of feeder layers or IL2 and their ability to be cloned and to grow in culture indefinitely.     

Upregulation of matrix metalloproteinase-9 in the cerebrospinal fluid of patients with acute Lyme neuroborreliosis

It was investigated (1) whether metalloproteinase-9 (MMP-9), MMP-3, and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1, the natural tissue inhibitor of MMP-9) are increased in the CSF of patients with Lyme neuroborreliosis and (2) whether macrophages can express MMP-9 when stimulated with Borrelia burgdorferi. Zymography showed MMP-9 activity in 26 of 31 (84%) CSF samples from patients with acute stage 2 Lyme neuroborreliosis, but not in 20 controls with non-inflammatory neurological disorders. Activity of MMP-2 was detected in all CSF samples in both patients with neuroborreliosis and controls, suggesting a constitutive release of MMP-2. Using enzyme linked immunosorbent assay (ELISA) MMP-3 (which can activate MMP-9) was detected in low concentrations in the CSF of 13 of 29 patients with neuroborreliosis, but not in controls. TIMP-1 was increased twofold in CSF samples from patients with neuroborreliosis in comparison with the controls. MMP-9 activity was induced in vitro in a mouse macrophage cell line (RAW 264.7) when stimulated with two different genospecies of B burgdorferi (B garinii, B afzelii ). This MMP-9 activity was reduced in a dose dependent manner when macrophages stimulated with B burgdorferi were coincubated with NF-kappaB SN50, a cell permeable peptide which inhibits the translocation of NF-kappaB into the nucleus of stimulated cells. The data show that (1) MMP-9 activity is present in the CSF of patients with neuroborreliosis, (2) macrophages stimulated with B burgdorferi are a possible source of MMP-9 increase, and (3) activation of NF-kappaB may play a part in the upregulation of MMP-9 by B burgdorferi.     

Correlation between neurologic disease and Borrelia burgdorferi antibodies in 800 paired serum/cerebrospinal fluid samples]

Paired serum and cerebrospinal fluid (CSF) samples from 800 patients of a neurological clinic were tested for antibodies to Borrelia burgdorferi. A flagellum enzyme-linked immunosorbent assay was used for antibody screening. All serum/CSF pairs with any elevated antibody response were also tested by a Western blot, a method for confirmation. 65 patients (8.1%) had serum IgG antibodies on ELISA screening and 22 of these (2.8%) were confirmed by Western blot. 20 patients (2.5%) had elevated antibody titers in CSF by ELISA and 12 of these (1.5%) reacted to the Western blot. Clinical features of Bannwarth's syndrome (BS) were present in 12 patients (1.5%) and four patients (0.5%) showed other manifestations of Lyme borreliosis. All patients with Bannwarth's syndrome were positive in serum by both methods and 10 had elevated antibody activity in the CSF proven by the two methods. The combination of a sensitive ELISA for screening and a sensitive and specific Western blot for confirmation reduces the number of positive results but is sensitive in detecting active disease.     

Cells secreting antibodies to myelin basic protein in cerebrospinal fluid of patients with Lyme neuroborreliosis.

An autoimmune response to myelin basic protein (MBP) has been proposed to participate in the development of the chronic neurologic manifestations that may accompany Borrelia burgdorferi-induced Lyme disease. Using an immunospot assay, we counted cells secreting antibodies to MBP. Anti-MBP IgG antibody-secreting cells were detected in CSF from eight of 13 consecutive patients with Lyme neuroborreliosis irrespective of stage of disease. The numbers were between 1/370 and 1/5,000 CSF cells (mean, 1/1,250 in the 13 patients). The highest numbers were encountered in two patients with severe signs of CNS involvement. The numbers decreased in parallel with clinical improvement after treatment. Anti-MBP IgG antibody-secreting cells were also observed in the CSF from patients with a variety of other inflammatory diseases of the nervous system, and their role in the development of tissue damage remains unsettled. Anti-MBP IgG antibody-secreting cells were not detected in the patients' blood, reflecting accumulation of this autoantibody response to CSF.     

T-cell phenotypic profiles in the cerebrospinal fluid and peripheral blood of multiple sclerosis patients.

Thirty-nine patients with clinically definite multiple sclerosis (MS) entered the study. Of 28 subjects with a relapsing-remitting course, 19 were classified in acute relapse, 9 in remission; 11 patients had a progressive course without remissions. Furthermore, 6 subjects with inflammatory neurological disease (IND), and 10 with non-inflammatory and non-neoplastic neurological disease (NIND) were investigated. We simultaneously studied cerebrospinal fluid (CSF) and peripheral blood (PB) T-, B- and NK-cell subsets, as defined by following monoclonal antibodies: anti-CD3, -CD4, -CD8, -CD19, -CD16, -HLA-DR and -IL-2-R. We found a significant increase of CD4+ T-cells compared with controls in CSF, with respect to PB, of MS patients, particularly in acute relapse. An increase of HLA-DR+ cell percentages in the CSF than in the PB in all MS groups, especially in attacks of MS but also in remission, was also observed, with a positive correlation between CD4+ T-cell and DR+ cell percentages both in the CSF as well as in the PB of relapsing MS patients. These findings, together with the increase of IL-2-R+ cells in the PB, particularly in relapsing MS, give further support for the presence of a systemic T-cell activation in MS.     

Lyme neuroborreliosis: cerebrospinal fluid contains myelin protein-reactive cells secreting interferon-γ

The mechanisms causing neurological manifestations and influencing the outcome in patients infected with the spirochete Borrelia burgdorferi are unknown. To study the involvement of autoimmune mechanisms in patients with Lyme neuroborreliosis (LN), ELIspot assays were used to determine the numbers of T lymphocytes which, upon stimulation with the myelin components myelin basic protein (MBP) and proteolipid protein (PLP) and two MBP peptides, responded by the secretion of interferon-γ (IFN-γ). LN patients had compared to patients with other neurological diseases and tension headache in CSF elevated numbers of MBP- and PLP-reactive T helper type 1 (Th1) like IFN-γ secreting cells at mean frequencies of about 1 per 1300 and 1 per 1500 CSF cells, respectively. Numbers were elevated up to 5 months after successful treatment of LN, and they did not correlate with clinical findings. Autoreactive T cells were not elevated in the LN patients' blood. These findings match the previously reported elevation of anti-MBP IgG antibody secreting cells exclusively in CSF in LN. The IFN-γ release by autoreactive cells and the secretion of autoantibodies in the CSF may have relevance for development and outcome of LN.     

Lyme borreliosis mimicking central nervous system malignancy: the diagnostic pitfall of cerebrospinal fluid cytology

We report two children with acute loss of neurological functions and signs of an increased intracranial pressure. Imaging techniques ruled out space occupying lesions, whereas CSF cytology indicated CNS involvement of a non-Hodgkin lymphoma in the form of abnormal lymphocytic pleocytosis with malignancy criteria fulfilling lymphoid cells. CSF protein electrophoresis and Borrelia burgdorferi serology revealed neuroborreliosis which was successfully treated with antibiotic therapy. The malignancy mimicking cytology is based on a blastoid transformation of B- and T-lymphocytes due to the antigenic stimulus of B. burgdorferi infection. Lymphoid cells in the CSF of a patient with acute or chronic neurological symptoms raise the differential diagnosis of inflammatory etiology versus CNS lymphoma. Monomorphism and higher quantity of the lymphoid cells point to CNS lymphoma. A lower quantity and polyclonal pattern of lymphoid cells associated with an elevated protein fraction caused by intrathecal immunoglobulin synthesis suggest an inflammatory etiology.