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Determination of matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF) to study blood–brain barrier impairment and immune cell migration in inflammatory neurological diseases recently became a matter of major interest. Regularly, MMP-9 was determined qualitatively or semi-quantitatively by zymography (gelatin gel electrophoresis) or quantitatively by enzyme immunoassay (EIA). As yet, it was not possible by either method to detect MMP-9 in CSF of controls (patients without pathologically increased CSF parameters). We developed an ultrasensitive two-side enzyme-linked immunosorbent assay (ELISA) which allows for the first time to measure reliably MMP-9 concentrations in CSF of controls. This ELISA uses a monoclonal as capture and a polyclonal as detector antibody. The detection limit of the assay is below 10 pg/ml and the assay range is 15–2000 pg/ml. Intra-assay precision is 2.5% for low and 3.7% for high, inter-assay precision is 11% for low and 10.7% for high values, respectively. The determination of the MMP-9 concentration in 50 control CSF gave the following results: range, 22–146 pg/ml; median, 76 pg/ml. The measurement of native and recombinant MMP-9 was carried out with three commercially available ELISAs, most widely employed in MMP-9 research, and compared to the newly developed one. All ELISAs recognize recombinant MMP-9 by factors of 5–20 less sensitively than native MMP-9.

Although multiple sclerosis (MS) is considered primarily as a demyelinating disease, neuronal damage is abundant and correlates with the neurological deficit. Therefore, we investigated the frequency and characteristics of human T cells specific for synapsin-a neuronal protein highly conserved among species. Synapsin specific T cell responses were detected at a frequency similar to that of MBP specific T cells in MS patients, one patient with acute demyelinating encephalomyelitis (ADEM) and controls. Long-term T cell lines specific for synapsin exhibited a CD3(+), CD4(+), CD8(-) phenotype and produced high amounts of tumor-necrosis-factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) after antigen specific stimulation, whereas lymphotoxin (LT), interleukin-4 (IL-4) and interleukin-10 (IL-10) were detectable in smaller quantities.

Matrix metalloproteinase-9 (MMP-9) was investigated by enzyme-linked immunosorbent assay (ELISA) and zymography in 111 paired CSF and serum samples from patients with various neurological disorders. In 20 patients with blood-brain barrier (BBB) impairment but normal CSF cell count, elevated levels of MMP-9 were not observed by ELISA measurement. Another 11 patients characterized in the same way, exhibited only slightly increased MMP-9 levels. In contrast, in 12 patients with intact BBB but elevated CSF cell count, MMP-9 was increased too. It was shown by the more sensitive zymography that MMP-9 increased if CSF cell count exceeded five cells per microl. Spearman rank statistics revealed that MMP-9 concentration in CSF correlated with CSF cell count (r=0.755; P

In the brain, S100 protein and neuron-specific enolase (NSE) are mainly found in glial cells and neurons, respectively. We investigated concentrations of S100 protein and NSE in cisternal cerebrospinal fluid obtained during implantation of foramen ovale electrodes in eight patients with temporal lobe epilepsy (TLE). In addition, the meningeal markers cystatin-C and beta-trace as well as total protein were measured. Patients with trigeminal neuralgia (TN) undergoing glycerol rhizotomy served as controls. S100 protein and NSE levels ipsilateral to the site of seizure onset were significantly higher than in TN. Contralateral TLE values were also markedly but not significantly elevated. The meningeal markers cystatin-C and beta-trace protein as well as total protein did not differ in TLE and TN. We conclude that interictal temporal lobe dysfunction corresponds with neuronal and glial marker elevations in the extracellular space and that site-specific elevations may predict the site of seizure origin biochemically.

Several studies have reported a positive correlation between levels of soluble adhesion molecules in serum or cerebrospinal fluid and cranial MRI activity. We performed a cross-sectional study in 46 patients with newly diagnosed MS and determined levels of soluble intercellular adhesion molecule-1 (sICAM-1) as well as vascular cell adhesion molecule-1 (sVCAM-1) in correlation to the number and area of gadolinium enhancing lesions on cranial magnetic resonance images (MRI). The data revealed a significant positive correlation between sVCAM-1 serum levels and gadolinium enhancing lesions. In addition, CSF to serum ratios for sICAM-1 and sVCAM-1 correlated to MRI activity. In patients with a single enhancing lesion (SEL) there was a negative correlation between the QsCAM and the distance of the SEL to the ventricles. As these adhesion molecules are stable and markers of disease activity in MS, we further investigated sVCAM-1 serum levels during treatment with interferon beta-1b (Betaferon®). Significant increases in serum levels for sVCAM-1 in patients receiving Betaferon were associated with a favourable treatment response after 1 year in 17 out of 19 patients and correlated to decreased MRI activity, whereas stable or reduced sVCAM-1 levels occured more often in non-responders (five out of six patients). Therefore it can be hypothezised that soluble adhesion molecules are released from cerebral endothelial cells as an early immunoregulatory activity of the immune system to reduce cellular traffic across the blood brain barrier and this is further enhanced by IFN-beta treatment.

The diagnostic significance of intrathecally synthesized IgG and virus-specific antibodies to measles, rubella and varicella-zoster (MRZ) in cerebrospinal fluid (CSF) remains controversial in cases of acute optic neuritis (AON). This study evaluates the prognostic value of baseline CSF and serum markers in AON, and correlates them with magnetic resonance imaging (MRI) and progression to multiple sclerosis (MS). Paired CSF and serum samples from 36 AON patients, 26 MS patients and 22 controls were analyzed for albumin, IgG, oligoclonal IgG (OI), MRZ antibodies, and blood-CSF barrier function; baseline MRI scanning of the head was also performed. The most sensitive parameter for detection of intrathecal inflammation in AON was OI (75%). Baseline MRI scans revealed abnormalities in 46% of the 28 patients with AON. Fifty percent of AON patients developed MS over the following 4 years. Ninety four percent of patients progressing to MS were positive for either OI, MRI or both. Of the AON patients initially positive for MRI and intrathecally-produced MRZ antibodies, 86% developed MS after 4 years. Only 17% of AON patients with negative results for OI and MRI developed MS. Six patients with abnormal OI but normal MRI progressed to MS. CSF and serum analyses, together with MRI, are the methods of choice for prognostic evaluation of patients with AON.

Although meninges represent a major site of biosynthesis, beta-trace protein (beta-trace) has not been studied in the cerebrospinal fluid (CSF) of meningitis patients. We measured beta-trace in lumbar CSF of normal controls (n = 27) and in patients with various neurological diseases (n = 92) by an immunonephelometric assay. The mean concentration of beta-trace in CSF of control patients was 16.6+/-3.6 mg/l. In bacterial meningitis (n = 41), CSF beta-trace was significantly decreased (8.7+/-3.9 mg/l; P< 0.001), whereas in spinal canal stenosis it was elevated (29.2+/-10.3 mg/l; P= 0.002). In viral meningoencephalitis (n = 12), beta-trace CSF concentrations were normal. Beta-trace concentrations remained below the normal range even after curing of bacterial meningitis, and normalisation of CSF leucocytes and blood-CSF barrier function. Beta-trace may be a useful tool for studying the pathophysiology of bacterial meningitis.

Choline acetyltransferase (ChAT) was partially purified from human placenta and brain. In order to raise monoclonal antibodies, Balb/c mice were immunized with a preparation from placenta or with a mixture of eight synthetic peptides that were deduced from the primary structures of porcine and human ChAT. Polyclonal antibodies were raised in rabbits against five synthetic peptides deduced from the amino acid sequence of human ChAT. The monoclonal and polyclonal antibodies were characterized by their ability to recognize ChAT in various immunoassays: immunoblot, enzyme-linked immunosorbent assay (ELISA), two-side ELISA and immunohistochemistry. With one exception all monoclonal antibodies recognized ChAT on immunoblots, some were particularly sensitive; one bound active ChAT in ELISA when used as capture reagent; most antibodies recognized immobilized ChAT in ELISA. Two monoclonal antibodies out of nine gave particularly excellent results in staining cholinergic neurons and fibers on sections from rat and primate brain. With the help of nine synthetic peptides it was possible to evaluate two major binding sites for the monoclonal antibodies on the ChAT molecule, comprising amino acids 167-189 and 57-76, respectively.

We performed a prospective study to correlate quantitative brain magnetic resonance imaging activity (gadolinium-diethylenetriaminepentaacetic acid enhancement) to cerebrospinal fluid and serum levels of soluble adhesion molecules in 46 patients with newly diagnosed multiple sclerosis (MS) and 30 control subjects with other diseases of the central nervous system. In all patients, magnetic resonance imaging of the brain and lumbar puncture were performed on the same day. In 32 (70%) of 46 MS patients, 8 (80%) of 10 patients with acute viral encephalitis, but none of the control subjects with noninflammatory diseases, gadolinium-enhancing lesions were detected. There was a significant correlation between the cerebrospinal fluid/serum ratios for soluble intercellular adhesion molecule-1 and soluble vascular cell adhesion molecule-1 as well as serum levels for both molecules and the area of gadolinium-enhancing lesions. No obvious correlation was observed between magnetic resonance imaging findings and cerebrospinal fluid cell count, protein concentration, or intrathecal immunoglobulin production. In patients with a single periventricular gadolinium-enhancing lesion (n = 16), we observed a strong negative correlation between the distance from the lateral ventricles and the cerebrospinal fluid/serum ratios for soluble intercellular adhesion molecule-1/albumin and soluble vascular cell adhesion molecule-1/albumin. These results suggest that intrathecal production of the two soluble adhesion molecules, as well as serum levels for soluble vascular cell adhesion molecule-1, in patients with MS reflect magnetic resonance imaging activity of typical periventricular lesions.

We analysed sera and cerebrospinal fluid (CSF) of 20 cases of human T-cell lymphotropic virus type (HTLV-I) associated myelopathy/tropical spastic paraparesis (HAM/TSP) from Brazil and Iran and as controls, 16 Brazilian HTLVI seronegative individuals afflicted with other neurological diseases. It was observed in the HAM/TSP patients that: 1) all had an inflammatory reaction within the central nervous system (CNS); 2) 95% (19/20) showed oligoclonal bands reflecting intrathecal IgG synthesis; 3) 85% (17/20) presented a local synthesis of HTLVI antibodies; 4) 35% (7/20) had a measurable immunoglobulin (Ig) synthesis within the CNS. The CSF parameters of the HAM/TSP were compared with the clinical data (age at onset, duration of disease and disability level). Our data prove that CSF analysis is important for the diagnosis of HAM/TSP. There is no association between the severity of the disease and CSF findings.

Paired samples of cerebrospinal fluid (CSF) and serum from 17 patients with human T-cell lymphotrophic virus I (HTLV-I)-associated myelopathy, 5 patients with multiple sclerosis and 11 controls with non-inflammatory disorders (migraine, idiopathic epilepsy and myelopathy of unknown aetiology) were examined by enzyme-linked immunosorbent assay for the presence of the 60-kDa soluble form of tumour necrosis factor receptor (sTNF-R). The results were compared with blood-CSF barrier function, cell count and the intrathecal synthesis of HTLV-I antibodies. No correlation could be demonstrated. High levels of sTNF-R were found in CSF of patients with HTLV-I-associated myelopathy and multiple sclerosis. In addition, intrathecal sTNF-R was also detected in the patients with non-inflammatory diseases, indicating that sTNF-R is definitively a normal constituent of CSF.

A soluble form of the intercellular adhesion molecule- (sICAM-1) was measured in paired cerebrospinal fluid (CSF)/blood samples from 123 patients with differenr neurological diseases. Mean levels of circulating ICAM-1 in the blod were mean ±SD = 423 ± 184.6 ng ml−1 (range 44–1115 ng ml−1). Considerable differences of sICAM-1 in the CSF of patients wre observed between disease groups. In accute bacterial maningitis, sICAM-1 levels as high as of the serum concentration were detected in the CSF (n = 24; mean ± SD = 33.0 ± 23.7 ng ml−1; range: 4.8–93.9 ng ml−1). These changes coincided with a sever blood-CSF barrier dysfunction as indicated by a high CSF/blood ration for albumin (mean ± SD = 46.7 ± 52.2; range: 16.8–249.3). In patients with polyradiculitits (n = 9; mean ± SD = 14.5 ± 11.9 ng ml−1; range: 2.6–43.7 ng ml−1) a similar convariation between the albumin and sICAM CSF/blood ratios was detected. In patients with multiple sclerosis (n = 9; mean ± SD = 5 ± 4.3; arange: 0–12.7 ng ml−1 or HIV infection with neurological symptoms (n = 18; mean ± SD = 4.9 ± 3.2; range; 1–11.9 ng ml−1) low levels of sICAM-1 were detected in the CSG associated with intact blood-CSF barrier function in most patients. Among 13 patients with viral meningitis, only four had detectable levels of sICAM-1 in their CSF (mean ± SD = 1.0 ± 1.5 ng ml−1; range: 0–3.7). Interestingly, in a groupof patients with non-inflammatory disorders of the CNS soluble ICAM-1 was present only in three out of 50 CSF samples despite indication for blood-CSF barrier dysfunction in most cases. Repeated lumbar punctures revealed that persistent high CSF/serum ratios for sICAM-1 were associated with poor outcome. In patients with inflammatory diseases of the CNS, a signoficant correlation was observed between CSF/serumratios for albumin and sICAM-1 ([ifn = 169; r = 0.67; P < 0.001), whereas no relation between these parameters was detected in patients with non-inflammatory diseases These results indicate that sICAM-1 is a reliable marker for an inflammatory process within non-inflammatory process within the CNS which is associated with blood-CSF barrier distrubance.

A recently developed immunocytochemical antigen-binding sandwich test for the identification of specific activated B lymphocytes was applied to cerebrospinal fluid cells and peripheral blood mononuclear cells in patients with acute neuroborreliosis. Discrimination of antigen-binding phagocytes was achieved by double staining with monoclonal antibodies. Specific activated B lymphocytes were much more numerous in cerebrospinal fluid than in blood, showing great interindividual differences. When intrathecal immunoglobulin production was present, the number of specific activated B lymphocytes was also high. The specificity of all activated B lymphocytes ranged from 10% to 60% and was higher in the acute stage than after treatment.

Intrathecally produced antibodies specific for the infectious agent can be shown by immunoblot. A quantification is practicable by titration. Densitometric evaluation of the immunoblot by a new technique, IDEA (immunoblot for densitometric estimation of antibodies), does not only render titration unnecessary, but also has the advantage of presenting the differentiated local immune response against individual antigens of the infectious agent. Preliminary studies for the densitometric evaluation were performed with a measles virus immunoblot which had been developed with a cerebrospinal fluid (CSF) sample of a patient suffering from multiple sclerosis containing high antibody titers. A peroxidase-labeled anti-immunoglobulin antibody had been added. The intensity of the color reaction on the membrane dependent on the antibody concentration follows a saturation kinetics comparable to the Michaelis-Menten kinetics in enzymology. Thus, indices for the intrathecal antibody synthesis of each individual viral antigen can be calculated by taking the permeability of the blood-CSF barrier into consideration. This method is illustrated in a patient with zoster meningoencephalitis.

An immunocytochemical method is presented by which individual proliferating T and B lymphocytes can be detected in the cerebrospinal fluid (CSF) of patients with inflammatory and neoplastic diseases of the central nervous system. The absolute numbers of proliferating T and B lymphocytes were approximately equal, so that only a minority of the strongly prevailing T-cell population proliferates in the CSF during the immune process. The highest proliferation (25.7%) was found within the small subset of activated, i.e. cytoplasmic immunoglobulin-containing, B lymphocytes. This provides further evidence that these cells are not the terminally differentiated plasma cells of classical cytology but progenitor cells capable of further proliferation. B-cell lymphomas could be easily identified. The detection of abnormally proliferating cell subsets could help in the differentiation of opportunistic infection from leukaemic infiltration.

Six patients with herpes simplex encephalitis were investigated. Antibody activities against different viral antigens were determined in serum and cerebrospinal fluid with an enzyme-linked immunosorbent assay after adjusting both fluids to identical immunoglobulin G concentrations. In serum, the strength of the antiviral reaction remained low and no qualitative changes became detectable. In cerebrospinal fluid, at first locally synthesized antibodies were directed against the same antigens as in serum. Ten days later, the intrathecal reaction increased with additional antibodies against at least two antigens. Once established, this expanded heterogeneity remained stable during the course of the disease.

β-Trace, a protein that represents a major constituent of human cerebrospinal fluid with unknown function has been purified to apparent homogeneity by gel chromatography and electrophoresis. After sodium dodecylsulfate electrophoresis on polyacrylamide gels (SDS-PAGE) and Western blotting, the N-terminal sequence (28 amino acids) has been determined. The high degree of idendity with the corresponding sequences of prostaglandin D synthetase from rat (68%) and human (93%) suggests a strong relationship if not identity with this enzyme. Thus, 30 years after its discovery β-trace might unravel as a well-known protein of the prostaglandin metabolism.

Transmigration of human granulocytes across a basal lamina equivalent was studied in vitro. Transwell® inserts were coated with Matrigel®, a reconstituted basement membrane. Granulocytes (2×10 6 ) were applied to the upper chamber. As chemoattractant interleukin-8 (IL-8; 25 ng/ml) was added to the lower chamber. After 1 h of migration, cells were counted in the lower chamber. Specific hydroxamate inhibitors of MMPs (BB-3103, Ro 31-9790) or of serine proteases (Pefabloc®, leupeptin) were added at various concentrations to both chambers before the start of migration. Additional experiments were performed with α 2 -macroglobulin, a natural inhibitor of MMPs and a monoclonal antibody which specifically blocks the activity of MMP-9. Migration of granulocytes through Matrigel could not be reduced significantly by any of the MMP inhibitors. A dose-dependent impairment of transmigration was only found with Pefabloc, however, this substance also induced severe morphological changes of the cells. The other inhibitor of serine proteases, leupeptin, did not influence migration at all.

Matrix metalloproteinase-9 (MMP-9) and its specific inhibitor, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1), were analysed by enzyme-linked immunosorbent assay (ELISA) and by zymography in serum and cerebrospinal fluid (CSF) of patients with amyotrophic lateral sclerosis (ALS). In contrast to patients with inflammatory diseases, MMP-9 levels were not elevated in CSF of ALS patients. In serum, however, compared to healthy donors, MMP-9 was significantly (p = 0.0003) increased up to levels as high as those of viral meningoencephalitis (VM) or bacterial meningitis (BM) patients. MMP-9 levels remained elevated during long-term observation of ALS patients. In the absence of an inflammatory response, the results indicate that the increase of MMP-9 in serum of ALS patients might be caused by upregulation of MMP-9 in denervated muscles or in degenerating peripheral nerves following motor neurone loss.

Beta-trace protein concentrations in cerebrospinal fluid (CSF) and serum from 113 patients with various neurological diseases and 65 controls were determined with a sensitive and specific immunonephelometric assay. In adult control patients, beta-trace concentrations were 14.6+/-4.6 mg/L in CSF and 0.46+/-0.13 mg/L in serum, that is, 32-fold higher in CSF. beta-trace levels in CSF correlated with age as well as with the albumin CSF/serum ratio (Q(Alb)), which is considered a measure for blood-CSF barrier function. The relationship between CSF beta-trace levels and elevated Q(Alb) values was studied in various neurological diseases with CSF protein increase. In spinal canal stenosis, CSF beta-trace (mean=29.5+/-10.5 mg/L) correlated positively with increasing Q(Alb) values. In bacterial meningitis, CSF beta-trace (mean=8.7+/-3.9 mg/L) remained invariant to changes of Q(Alb) values. In Guillain-Barre syndrome, CSF beta-trace (mean=14.4+/-6.8 mg/L) was below the Q(Alb)-dependent reference range. In multiple sclerosis and viral meningoencephalitis, beta-trace levels were within the reference range. Beta-trace concentration in CSF can be used in conjunction with Q(AlB) to distinguish between different neurological pathologies associated with CSF protein increase.

Serum concentrations of two macrophage-derived calcium-binding proteins, MRP-8 and MRP-8/14, were studied in 28 patients with relapsing multiple sclerosis (MS). Serum levels were determined with a commercially available sandwich ELISA and the one-tailed Mann-Whitney U test was used for statistical analysis. Median serum levels of MRP-8/14 were significantly higher in MS patients (5150 ng/ml) compared to 26 healthy controls (1482 ng/ml) and significantly higher in MS patients within an acute relapse (6690 ng/ml) compared to MS patients with stable disease (3050 ng/ml). MRP-8 levels were not elevated in MS patients. These results may indicate an early activation of macrophages in the formation of demyelinating MS plaques. In addition, increased serum levels of MRP-8/14 may prove to be a useful paraclinical disease activity parameter in MS patients.

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

Cerebrospinal fluid (CSF) and serum of 17 patients with HAM/TSP (HTLV-I associated myelopathy/ tropical spastic paraparesis), six with multiple sclerosis and six with idiopathic epilepsy (non inflammatory control) from Brazil were analysed for the presence of intrathecal synthesis of virus-specific antibodies against measles, rubella, varicella zoster virus and herpes simplex virus by enzyme-linked immunosorbent assay (ELISA). All HAM/TSP and multiple sclerosis cases had an intrathecal immune response (oligoclonal IgG). In HAM/TSP, only 1/17 case showed a polyspecific intrathecal immune response against measles and rubella virus. In multiple sclerosis, specific antibodies against measles and rubella (MRZ response) were observed in all patients but not in the control with idiopathic epilepsy. The diagnostic and theoretical relevance of mono- and polyspecific immune responses is discussed for these chronic neurological diseases.

Bei der progressiven multifokalen Leukoenzephalopathie (PML) handelt es sich um eine sehr seltene, subakut demyelinisierende Erkrankung des zentralen Nervensystems (ZNS). Etwa 2 % bis 7% aller mit dem humanen Immundefizienzvirus (HIV) Infizierten entwickeln eine PML. Anhand kernspintomographisch nachweisbarer, nicht Kontrastmittel anreichernder, in der T2-Wichtung hyperintenser und in der T1-Wichtung hypointenser Lasionen uberwiegend der weisen Substanz last sich eine klinisch wahrscheinliche Diagnose stellen (Whiteman et al., 1993). Eine eindeutige Abgrenzung von einer HIV Leukenzephalitis/Enzephalopathie (HIVE), zerebralen Toxoplasmose, einem malignen Lymphom oder Hirntumoren ist dagegen nur bioptisch moglich. Da selbst die Biopsie nur eine Sensitivitat von etwa 61 % (Whiting et al. 1992) bis 96 % (Levy et al. 1992) aufweist, ist ein nicht-invasives Verfahren zum Virusnachweis dringend erforderlich. Mittels der Polymerase-Ketten-Reaktion (PCR) steht ein hochempfindliches und spezifisches Verfahren zur Verstarkung virusspezifischer Nukleinsauren zur Verfugung.

Using polymerase chain reaction (PCR), 34 cerebrospinal fluid (CSF) samples from 28 patients with progressive multifocal leukoencephalopathy (PML) were analyzed. As controls, 116 samples were evaluated from 82 human immunodeficiency virus type 1 (HIV-1)-infected patients and 1 HIV-1-negative patient. Of the HIV-1-positive patients, 23 had cerebral toxoplasmosis, 10 had HIV leukoencephalopathy, and 49 had other neurologic complications. Detection of JC virus (JCV) DNA in CSF was increased 10-fold by the addition of carrier DNA before phenol-chloroform-isoamyl alcohol extraction. The primer pair JC 26/29, from the VP1/large T region, had a limit of detection of 10(5) JCV DNA molecules/100 microL. The primer pair JC 36/39, located in the large T gene region, had a 100-fold lower limit of detection. With JC 26/29, the sensitivity was 43% (12/28) and specificity was 100%. Using JC 36/39, sensitivity increased to 82% (23/28), and false-positive results were not observed. Diagnosis of PML is greatly aided by PCR analysis of CSF.

The clinical, neuroradiological, and cerebrospinal fluid findings of a case with acute diffuse leukoencephalitis, a demyelinating disease associated with human immunodeficiency virus infection of the brain, is reported. The patient presented with acute tetraparesis as the primary manifestation of a previously symptom free HIV infection. Cerebrospinal fluid analysis showed enhanced inflammatory abnormalities with high concentrations of P24 antigen. MRI showed diffuse white matter hyper-intensities in both hemispheres. In the follow up over 22 months, the neurological deficits disappeared after antiretroviral treatment in good correlation with improvements in MRI as well as in inflammatory cerebrospinal fluid abnormalities.

Five young patients are described with biopsy-proven acute demyelinating disease. Two cases are classified as Schilder's disease, a particular childhood form of multiple sclerosis (MS) with atypical clinical manifestation, normal or atypical CSF-findings and large bilateral lesions in magnetic resonance imaging (MRI). Two further cases presented with a fulminant bout of MS with typical clinical picture and CSF-findings; they are classified as Marburg's disease. The last case was an acute second bout of classical MS. The biopsy seemed to be justified in 4 cases, but unnecessary in the last case. Localized proton magnetic resonance spectroscopy (MRS) performed in one case allowed us to examine the focal cerebral abnormalities directly and non-invasively. The spectra revealed a pattern typical of acute demyelination suggesting potential for a replacement of biopsy in the future.

The antibody specificity index (ASI) indicates the cerebrospinal fluid (CSF)/serum difference of antibody amounts per weight unit IgG (normal less than 1.5). It has proven to be the most sensitive inflammation parameter in CSF analysis so far, more sensitive than the Western blot, the "oligoclonal" response, and the empirical differentiation of CSF immunoglobulins. By this diagnostic criterion, several benign viral meningitis cases were found to be caused by the varicella/zoster virus. The diagnostic relevance of local zoster antibody synthesis was greatest in ganglionitis cases, e.g., in zoster oticus sine herpete (facial paresis) and acute radicular syndromes of the elderly. The diagnostic significance of the local immune response against measles, rubella, and zoster antigens (MRZ response) was ascertained further. Together with oligoclonal gamma-globulin fractionation, there is now only 1 out of 100 multiple sclerosis (MS) patients left who has been found to have a normal CSF.

An ELISA procedure is described for the quantification of intrathecally synthesized immunoglobulin G antibodies to human immunodeficiency virus (HIV) antigens. Recombinant p17, p24, endonuclease (END), reverse transcriptase (RT), a peptide from the transmembrane region of gp41 (ENV80) and a fusion protein containing HIV-1 and HIV-2 epitopes were compared with a commercially available ELISA. Using a reference serum, antibodies in serum and cerebrospinal fluid (CSF) to all of the antigens could be measured quantitatively in a reliable and reproducible fashion. Despite the fact that the titer varied up to 10(5)-fold between CSF and serum, interassay variability ranged from 3.87% for p17 to 8.41% for RT and intra-assay variability varied from 3.9% +/- 1.2% for p17 to 14.3% +/- 3.9% for the commercial ELISA. Antibody specificity indices (ASI) obtained by relating CSF/serum titers with reference to the corresponding IgG concentrations can be used to detect intrathecal synthesis of virus specific antibodies.

Early diagnosis of acute cerebral ischaemia is still unsatisfactory, because X-ray computed tomography (CT) does not reveal the site and extent of hypoperfusion within the first 24 h. Single photon emission computed tomography (SPECT) using 99mTc-hexamethylpropylene amine oxime (HMPAO) may offer earlier information, since the distribution of HMPAO follows the actual cerebral perfusion pattern. We therefore investigated 53 patients suffering from acute cerebral ischaemia (10 with transient ischaemic attacks, 9 with prolonged ischaemic reversible neurological deficits, 34 with completed stroke). SPECT and CT examinations were performed on days 1, 3, and 14. On day 1, SPECT revealed hypoperfused areas in 42 patients, whereas CT showed hypodensities only in 5. The sensitivity of SPECT was higher in cortical compared with subcortical ischaemia. In patients suffering from reversible neurological deficits SPECT normalized in the follow-up, corresponding to clinical improvement. In completed stroke, SPECT demonstrated variable perfusion patterns with hypo-, normo-, and hypoperfused areas on day 3 and especially on day 14. In contrast to CT, HMPAO SPECT leads to early diagnosis of cerebral ischaemia, in particular within the cerebral cortex.

Applying the immunoblot technique a sensitive and specific method was developed for the detection of intrathecally synthesized antibodies against individual specific proteins that are antigens of various infectious agents causing encephalitis. Paired serum and cerebrospinal fluid (CSF) samples from five patients with herpes virus infections of the central nervous system (CNS) (three herpes simplex virus encephalitis, one varicella zoster virus encephalitis, one zoster ganglionitis) were investigated for the presence of locally produced IgG against the electrophoretically separated antigens of herpes simplex virus (HSV), varicella zoster virus (VZV) and human cytomegalovirus (HCMV), as well as for IgM antibodies in one case of HSV encephalitis. In two cases (HSV encephalitis and VZV encephalitis) four and one antibody, respectively, were found that were synthesized intrathecally only. In the other cases the patterns of sera and CSF antibodies were similar, the CSF antibodies showing an all-over stronger reaction, at identical IgG concentrations. IN contrast to the conception of a ‘limited heterogeneity’ of intrathecal antibody synthesis in encephalitis, we thus found an ‘ expanded heterogeneity’ of the intrathecally sythesized antibodies in comparison to the corresponding serum antibodies.

Multiple sclerosis lesions may occur predominantly in the hemispheric white matter and cause various psychiatric disorders such as remitting-relapsing endoform or exogenous psychosis, organic personality alterations and dementia. Nineteen patients suffering from this encephalitic form of multiple sclerosis as diagnosed by characteristic CSF immunoglobulin findings are analysed according to established psychopathological criteria. All cases began with psychiatric symptoms and neurological signs were either absent or overlooked. Several patients developed typical encephalomyelitic symptoms in successive relapses, but other remained with psychiatric disorders over many years. Only four patients had retrobulbar neuritis, but seven suffered from epileptic seizures. The humoral immune response was characterized by a strong dominance of IgG and a local synthesis of polyspecific antibodies against measles, rubella and varicella/zoster virus. The mononuclear CSF pleocytosis was comparatively marked with cell counts up to 180/microliter.

An immunocytochemical technique allowing repeated use of antisera is applied to identify immunoglobulin-containing cells (ICC) of the IgG, IgA, and IgM class in the cerebrospinal fluid (CSF) of 298 patients with various neurological disorders. The demonstration of ICC in the CSF is highly indicative of an inflammatory disease (p less than 0.0001; Chi-square test). In the group of noninflammatory disorders ICC are only found in three cases of lymphomas, two dysgerminomas, and one glioblastoma. ICC of all classes are seen in acute viral and bacterial infections of the CNS including tick-borne meningopolyneuritis Bannwarth. IgG-positive ICC predominate in chronic inflammatory disorders like multiple sclerosis and HIV encephalitis. In HIV-positive patients IgA- or IgM-positive cells are strongly indicative of an opportunistic infection of the brain. Persistent high levels of ICC in three patients with bacterial meningitis are associated with a fatal outcome.

To establish a reliable procedure for the early detection of central nervous system involvement in HIV infection, paired cerebrospinal fluid and serum samples of 59 patients were analysed. Fifteen were HIV antibody positive without clinical symptoms (stage I), 12 had lymphadenopathy syndrome or AIDS-related complex (stage II), and 32 had AIDS (stage III). Intrathecal synthesis of HIV antibodies was determined by a modified ELISA. Antibodies in CSF and serum were evaluated at identical immunoglobulin G levels to correct for the actual blood-CSF-barrier permeability. A CSF/serum quotient above 1.5 is indicative of intrathecal antibody synthesis, which was found in 47% of the patients in stage I, 67% in stage II, and 84% in stage III. These findings indicate an early and frequent invasion of the CNS.

Intrathecal antibody responses to HIV were investigated by a highly sensitive immunoblot assay. Serum and CSF specimens were tested for reactivity with the recombinant HIV gag proteins p15, p17 and p24 and with the recombinant transmembrane protein p41. Autochthonous production of virus-specific IgG to one or more HIV structural proteins was seen in 8 of 10 asymptomatic seropositive subjects, in 3 of 4 men with AIDS-related complex, and in 9 of 13 patients with AIDS. These results were consonant with an elevated CSF/serum antibody ratio to total HIV antigen. The high frequency of local HIV-specific antibody synthesis in seropositive individuals without related clinical symptoms indicates an early involvement of the CNS in HIV infections.

Cells containing immunoglobulins G, A, and M were evaluated in paired cerebrospinal fluid (CSF) and peripheral blood (PB) samples. These were obtained from 12 patients with bacterial meningitis, 14 patients with viral meningitis, 6 cases of lymphocytic meningoradiculitis (LMR), 10 cases of multiple sclerosis (MS), 6 cases of herpes zoster ganglionitis and 27 patients with non-infectious disorders of the CNS. PB cells from 20 healthy donors served as controls. Using alkaline phosphatase (AP)-conjugated antibodies to human immunoglobulin (Ig) G, A, and M in a carrageenan solution it was possible to demonstrate repeatedly intracytoplasmic Igs over more than 1 year without any detectable loss of specificity and staining intensity. Immunoglobulin-containing cells (ICC) could be detected in the CSF of 96% of patients with inflammatory diseases of the central nervous system (CNS) or with MS but not in the control cases.

Sixteen out of eighteen meningeal carcinomas (89%) secreted carcinoembryonic antigen (CEA) into the cerebrospinal fluid, where it could be quantified separately from the portion originating from the circulating blood. The discrimination of both fractions was performed according to an empirical approach analogous to the immunoglobulins. Only 47% of the intraparenchymal carcinomas released CEA into the CSF compartment and it is possible that the extra-cellular space of these tumour sites does not communicate with the free CSF space. Extradural metastases may cause an impairment of the blood-CSF barrier via restrictions of the CSF fluid turnover, but the dura remains impermeable for the tumour marker. Seven out of 54 primary brain tumours (13%) released carcinoembryonic antigen into the cerebrospinal fluid.

A method is presented that allows the immunocytochemical detection of varicella zoster virus antigen-binding cerebrospinal fluid cells in zoster ganglionitis. Antigen-binding cells were found only in patients suffering from this disease. The technique is sensitive, specific, inexpensive and relatively fast, and is potentially applicable to other inflammatory central nervous system diseases with immunoglobulin-containing cells (ICC) in CSF. The detection of antigen-binding CSF cells may represent a very early diagnostic test comparable with the early IgM antibodies of the systemic immune response.

A patient is described who developed complex partial seizures with secondary generalization 3 years after a severe viral encephalitis with a CT and EEG identified lesion in the left insular cortex and its surrounding structures. When the seizures first occurred CT and MRI as well as repeated interictal conventional EEG recordings were entirely normal. Single photon emission computed tomography (SPECT), however, revealed an area of increased 99mTc-hexamethyl propyleneamine oxime (HMPAO) uptake in the left insular cortex. After anticonvulsive therapy the seizures and the SPECT findings disappeared. 99mTc-HMPAO SPECT is a highly sensitive method for the demonstration of functional alterations in brain tissue. It can improve diagnosis of epilepsy and may provide additional information to monitor anticonvulsive therapy.

We found that eight of 22 patients with meningeal carcinomatosis from different primary tumors produced local IgG in the CSF, as indicated by elevated IgG index and/or oligoclonal IgG subfractions. Local IgG production, when present, appears in an early stage of the leptomeningeal manifestation and remains detectable over extended observation periods. In autopsied cases with local IgG production, we observed numerous perivascular round-cell infiltrates containing plasma cells and large lymphocytes within the leptomeningeal tumor tissue. After incubation with immunoperoxidase, only these cells showed IgG-specific staining, indicating the site of local IgG production.

Combined medical, neurological, and serological investigations were carried out in 59 patients infected with human immunodeficiency virus (HIV). In stage I clinical and neuropsychiatric testing did not reveal evidence for HIV encephalitis as diagnosed by local antibody production in CSF. Neuropsychiatric abnormalities, brain atrophy, memory and cognitive impairment reliably indicated HIV encephalitis in later stages. The commonest symptoms were cerebellar and brainstem signs, followed by dementia. Epileptic fits and hemipareses always were associated with cerebral toxoplasmosis. A polyneuropathy was frequently found but other causes have to be considered.