β-N-Acetylhexosaminadases in human cerebrospinal fluid and serum of patients with multiple sclerosis

β-N-Acetylhexosaminidase activity and isoenzyme have been investigated in normal human cerebrospinal fluid and that of patients with multiple sclerosis. β-N-acetylhexosaminidase activity in normal cerebrospinal fluids has been resolved into five components. The major component was in a form that eluted from DEAE cellulose at the same salt concentration as hexosaminidase As, the isoenzyme previously identified in human serum. Cerebrospinal fluid from patients exhibited a different isoenzyme profile, showing a remarkable increase in a form having a pI which was more acidic than that of As. These changes have a potential use in the diagnosis and further biochemical characterization of multiple sclerosis.     

Detection of myelin basic protein in cerebrospinal fluid

Radioimmunoassay for myelin basic protein in cerebrospinal fluid is commonly used as a biochemical marker of demyelination in multiple sclerosis patients. A sensitive enzyme-linked immunosorbent assay for myelin basic protein has been recently developed, which can make a clinical evaluation of myelin basic protein in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. Most multiple sclerosis patients with acute exacerbation had markedly high myelin basic protein. Longitudinal studies of multiple sclerosis patients showed that myelin basic protein in CSF increases rapidly in agreement with acute relapse and then rapidly declines and disappears. Significantly higher cerebrospinal fluid myelin basic protein levels in human T-cell lymphotropic virus Type I-associated myelopathy/tropical spastic paraparesis patients were also detected. This enzyme-linked immunosorbent assay system can be used routinely to measure myelin basic protein in cerebrospinal fluid as a useful diagnostic indicator, not only for central active demyelination as in multiple sclerosis but, also for spinal cord demyelination as in human T-cell lymphotropic virus Type I-associated myelopathy/tropical spastic paraparesis.     

Cystatin C, alias post-gamma-globulin: a marker for multiple sclerosis?

Cystatin C, alias post-gamma-globulin or gamma-trace protein, has been shown to be a potent inhibitor of cysteine proteinases; this protein is normally present in different biological fluids, but particularly so in cerebrospinal fluid. The concentration of cystatin C was determined by radial immunodiffusion in cerebrospinal fluid from patients affected with multiple sclerosis, patients affected with various neurological diseases and in controls; it was also determined in brain tissue from 2 patients affected with multiple sclerosis and 3 control brains. Cystatin C cerebrospinal fluid levels were undetectable or depressed in many multiple sclerosis cases and the median value differed significantly from the control one. Its low concentration in multiple sclerosis suggests that the regulation of cysteine proteinases is impaired in this disease; hence enhanced activity of cysteine proteinases could initiate, or increase the breakdown of myelin. Although it is perhaps a little premature to consider cystatin C as a marker for multiple sclerosis, this protein is nevertheless associated to demyelination; consequently its biochemical assay in cerebrospinal fluid is recommended as a complementary diagnostic tool.     

Human kallikrein 6 as a biomarker of alzheimer's disease

Background: Alzheimer’s disease (AD) is a major cause of dementia in the elderly. It is generally difficult to diagnose accurately early AD. A few biomarkers, including τ protein and amyloid β-42, are now used as aids for diagnosis and monitoring of AD. Our aim was to examine the possible use of cerebrospinal fluid, blood and tissue, and human kallikrein 6 (hK6) concentration as a marker of AD.

Methods: We have used a highly sensitive and specific immunofluorometric procedure for measuring hK6. We measured hK6 in tissue extracts from AD brain or normal individuals, in cerebrospinal fluids of AD patients or normals and in whole blood of AD patients and normals and compared the findings. We have used ten pairs of AD/normal controls in all cases.

Results: We found that hK6 concentration is tissue extracts from AD brain were approximately twofold lower than extracts from normal controls. Further, we found that cerebrospinal fluid hK6 concentration is approximately a threefold increase, in comparison to cerebrospinal fluid controls (p = 0.001). We have also found that the whole blood hK6 concentration in AD patients is about ten times higher than hK6 concentration in normal controls (p = 0.002). We have immunohistochemically localized the expression of hK6 in epithelial cells of the chorioid plexus.

Conclusions: This is the first report describing significant elevations of cerebrospinal fluid and plasma and whole blood hK6 concentration in AD patients, in comparison to controls. These data suggest that hK6 may constitute a new biomarker for diagnosis and monitoring of AD.     

The effect of corticosteroids on serum and cerebrospinal fluid nitric oxide levels in multiple sclerosis.

Multiple sclerosis is a disease characterized by perivascular infiltrates and demyelination of the white matter in the central nervous system. In this study, we compared the serum and cerebrospinal fluid nitric oxide levels before and after methylprednisolone therapy, and during remission period, and investigated the relationship of nitric oxide to the activity of multiple sclerosis. Cerebrospinal fluid and serum nitric oxide levels were measured blind as nitrite plus nitrate, using the nitrate reductase and Griess reaction method in 20 patients with multiple sclerosis before and after corticosteroid therapy, and during remission period, and in 20 control subjects. Mean cerebrospinal fluid and serum nitric oxide levels were highest in the pretreatment group and lowest in the control group. There was no correlation with nitric oxide levels in these two groups. Although corticosteroid therapy did not have any great effect on Expanded Disability Status Scales, it led to a decrease in nitric oxide levels. The possible cause of this might be the inhibition of nitric oxide synthesis by methylprednisolone, or a decrease in multiple sclerosis activity. We conclude that serum or cerebrospinal fluid nitric oxide levels do not reflect the activity in multiple sclerosis.     

Lecithin cholesterol acyltransferase in human cerebrospinal fluid: reduced level in patients with multiple sclerosis and evidence of direct synthesis in the brain

Summary Several studies suggest that apolipoproteins and the low-density lipoprotein receptor are implicated in lipid transport in the brain. Given that cerebrospinal fluid has been reported to contain cholesteryl esterifying enzyme, and that lipid metabolism in the brain is abnormal in subjects with multiple sclerosis, we examined the cerebrospinal fluid from eight control subjects with a normal cerebrospinal fluid IgG index, and without active demyelinating disease, and from eight subjects (6 were diagnosed as having multiple sclerosis) with an increased IgG index and the presence of oligoclonal banding in the cerebrospinal fluid, for the presence of the enzyme lecithin cholesterol acyltransferase and apolipoprotein(a). None of the subjects demonstrated imapairment of their blood-cerebrospinal fluid barrier, as estimated by the cerebrospinal fluid/serum quotient of albumin. Lecithin cholesterol acyltransferase was detected in the cerebrospinal fluid of all control subjects, being 0.12±0.06 μg/ml (mean±SD) or about 2.2% of that in serum (5.4±1.4 μg/ml). The cerebrospinal fluid lecithin cholesterol acyltransferase index was 5.2±2.5, very similar to the cerebrospinal fluid index of apolipoprotein E, a protein known to be synthesized in the brain. Since lecithin cholesterol acyltransferase mRNA is also expressed in the brain, we can conclude that the protein is synthesized and secreted in the brain. The cerebrospinal fluid concentration of lecithin cholesterol acyltransferase in the subjects with active demyelinating disease or multiple sclerosis was only about one-half of that found in control subjects (0.06±0.02 μg/ml). Lipoprotein(a) concentration was below the sensitivity of the enzyme immunoassay used for the measurement (<40 ng/ml) in cerebrospinal fluid from both control subjects and those with demyelinating disease or multiple sclerosis.     

Lecithin cholesterol acyltransferase in human cerebrospinal fluid: reduced level in patients with multiple sclerosis and evidence of direct synthesis in the brain.

Several studies suggest that apolipoproteins and the low-density lipoprotein receptor are implicated in lipid transport in the brain. Given that cerebrospinal fluid has been reported to contain cholesteryl esterifying enzyme, and that lipid metabolism in the brain is abnormal in subjects with multiple sclerosis, we examined the cerebrospinal fluid from eight control subjects with a normal cerebrospinal fluid IgG index, and without active demyelinating disease, and from eight subjects (6 were diagnosed as having multiple sclerosis) with an increased IgG index and the presence of oligoclonal banding in the cerebrospinal fluid, for the presence of the enzyme lecithin cholesterol acyltransferase and apolipoprotein(a). None of the subjects demonstrated impairment of their blood-cerebrospinal fluid barrier, as estimated by the cerebrospinal fluid/serum quotient of albumin. Lecithin cholesterol acyltransferase was detected in the cerebrospinal fluid of all control subjects, being 0.12 +/- 0.06 microgram/ml (mean +/- SD) or about 2.2% of that in serum (5.4 +/- 1.4 micrograms/ml). The cerebrospinal fluid lecithin cholesterol acyltransferase index was 5.2 +/- 2.5, very similar to the cerebrospinal fluid index of apolipoprotein E, a protein known to be synthesized in the brain. Since lecithin cholesterol acyltransferase mRNA is also expressed in the brain, we can conclude that the protein is synthesized and secreted in the brain. The cerebrospinal fluid concentration of lecithin cholesterol acyltransferase in the subjects with active demyelinating disease or multiple sclerosis was only about one-half of that found in control subjects (0.06 +/- 0.02 microgram/ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

A single-step solid phase radioimmunoassay for quantifying human carbonic anhydrase I and II in cerebrospinal fluid

A single-step solid phase radioimmunoassay was developed to detect human carbonic anhydrase (CA) isoenzymes I (CA I) and II (CA II) in cerebrospinal fluid (CSF). The assay is capable of routinely detecting both isoenzymes at ng levels compared to the μg levels of the traditional catalytic methods, which failed to demonstrate any CA activity in CSF. When the values of immunoreactive CA II in CSF were corrected for blood contamination (the CA I/CA II ratio of blood was about 7.9), the amount of brain tissue originated CA II could be calculated. The CA II values in CSF samples from 13 patients with multiple sclerosis were higher than those in CSF samples from 11 patients with various peripheral neurological disorders. Since CA II has been specifically localized to oligodendrocytes and myelin, our preliminary results suggest the possibility of CA II leakage from oligodendrocytes and myelin into CSF in demyelinating diseases.     

Contrast medium causes the apparent increase in β-endorphin levels in human cerebrospinal fluid following brain stimulation

Levels of β-endorphin immunoreactivity in cerebrospinal fluid were measured in 12 chronic pain patients undergoing the surgical implantation of an electrode into the periventricular gray matter. Cerebrospinal fluid fractions were collected following placement of a cannula into the third ventricle, following injection of metrizamide contrast medium into the ventricles, following implantation of the electrode, and following electrical stimulation. A second set of samples was collected on a non-surgical day before and after stimulation.

Levels of β-endorphin immunoreactivity increased significantly from baseline levels to post-electrode implantation in one group of patients, but no significant change was seen following the onset of stimulation. Immunoreactivity increased significantly following metrizamide injection in a second group and was still elevated, in comparison to baseline, following electrode placement, but no increase was seen following the onset of stimulation. Levels of immunoreactive β-endorphin did not increase in either group after stimulation on a post-surgical day, despite consistent reports of pain relief. Addition of metrizamide or a related contrast medium, iothalamate meglumine (Conray) to the β-endorphin radioimmunoassay revealed that both compounds interfered with antigen-antibody binding and also quenched the gamma radiation emitted by iodinated peptide ligands. Due to these combined effects, the contrast media alone produced results similar to those of the β-endorphin standard. Moreover, similar observations were made when contrast media were incorporated into radioimmunoassays for met-enkephalin, dynorphin and cholecystokinin octapeptide.

These findings indicate that increased levels of β-endorphin in cerebrospinal fluid are not directly associated with patient report of pain relief following periventricular gray stimulation. This study also suggests that the previously reported association between periventricular gray stimulation and elevated levels of β-endorphin immunoreactivity in ventricular cerebrospinal fluid are artifactual due to the interaction of contrast media with the radioimmunoassay.     

The activity of 2',3'-cyclic nucleotide 3'-phosphohydrolase in human cerebrospinal fluid.

A method for the determination of cyclic nucleotide phosphatase (CNP) activity in cerebrospinal fluid is presented. In normal CSF the activity of CNP is very low. Comparing CSF from patients with multiple sclerosis to control CSF no significant difference is found, although a small proportion of MS patients has an elevated has an elevated CNP activity in their CSF. It is postulated that similar to other substances the CNP activity in CSF probably does not derive from the central nervous system.     

Low molecular weight thiol content in glutathione synthetase-deficient human fibroblasts

The activity and the kinetic properties of glutathione synthetase and the concentrations of non-protein bound thiols of the γ-glutamyl cycle were measured in 11 human fibroblast cell strains. Six of these strains were derived from patients suffering from 5-oxoprolinuria, a recessive genetic disease characterized by a deficiency in glutathione synthetase; the other cell strains were derived from healthy heterozygous or homozygous relatives of the patients. The glutathione synthetase activities of homozygous deficient strains were 1/3 of control values while those of heterozygous strains were 2/3 of control values. The total thiol concentration was lower in only 3 of the 6 deficient homozygotes and that of glutathione (GSH) was lower in only 4 of the 6 deficient homozygotes. This lower GSH level was at least partly offset by an accumulation of γ-glutamylcysteine, a precursor of GSH, which is almost completely absent from control cells. The total quantities of thiols and GSH in plateau phase cells were about 50% and 30% respectively of the levels in growth phase cells. Approximately 80% of the GSH was in the reduced form in both quiescent and growing cells.     

Lysosomal enzymes in motor neurone disease and multiple sclerosis

In nine patients with motor neurone disease the activity of acid phosphatase in cerebrospinal fluid (CSF) was higher (P<0.005) than in nine controls. The activity of this enzyme in CSF from 18 patients with multiple sclerosis did not differ from control values. The activity of arylsulphatase in CSF was the same in the motor neurone, multiple sclerosis and control groups. In the serum, the activities of the two enzymes were similar in the three groups of patients.     

Broader Epstein–Barr virus–specific T cell receptor repertoire in patients with multiple sclerosis

Epstein–Barr virus (EBV) infection precedes multiple sclerosis (MS) pathology and cross-reactive antibodies might link EBV infection to CNS autoimmunity. As an altered anti-EBV T cell reaction was suggested in MS, we queried peripheral blood T cell receptor β chain (TCRβ) repertoires of 1,395 MS patients, 887 controls, and 35 monozygotic, MS-discordant twin pairs for multimer-confirmed, viral antigen–specific TCRβ sequences. We detected more MHC-I–restricted EBV-specific TCRβ sequences in MS patients. Differences in genetics or upbringing could be excluded by validation in monozygotic twin pairs discordant for MS. Anti–VLA-4 treatment amplified this observation, while interferon β– or anti-CD20 treatment did not modulate EBV-specific T cell occurrence. In healthy individuals, EBV-specific CD8⁺ T cells were of an effector-memory phenotype in peripheral blood and cerebrospinal fluid. In MS patients, cerebrospinal fluid also contained EBV-specific central-memory CD8⁺ T cells, suggesting recent priming. Therefore, MS is not only preceded by EBV infection, but also associated with broader EBV-specific TCR repertoires, consistent with an ongoing anti-EBV immune reaction in MS.     

Myelin basic protein, oligoclonal bands, and IgG in cerebrospinal fluid as indicators of multiple sclerosis

There currently are three clinical laboratory procedures for use with cerebrospinal fluid that assist in the diagnosis of multiple sclerosis: measurement of myelin basic protein and IgG, and demonstration of an oligoclonal band. We compared characteristics of these procedures, using CSF samples from 166 patients identified as having (54 patients) or not having (112 patients) multiple sclerosis. We find that oligoclonal band demonstration is the most useful single test in helping to establish the presence of multiple sclerosis; IgG quantitation is the least helpful. Myelin basic protein should be quantitated for following the activity of multiple sclerosis; it may be applied only selectively in the context of screening. The incidence of false-positive results reinforces the view that the diagnosis of multiple sclerosis must be made in clinical context. These laboratory procedures are not suitable for use as screening tests.     

Beta-adrenoceptors in cardiac disease

The human heart contains both β₁ and β-₂-adrenoceptors; both mediated positive inotropic and chronotropic effects. In chronic heart failure, β-adrenoceptor number is reduced, presumably, by down-regulation by endogenous noradrenaline which is elevated due to increased sympathetic activity. Since the human heart contains only a few spare receptors for β-adrenoceptor-mediated positive inotropic effects and the amount of spare receptors declines in chronic heart failure, it is not surprising that the reduced β-adrenoceptor number is accompanied by decreased contractile responses to β-adrenoceptor agonists (including endogeneous catecholamines), and the extent of decrease in maximal inotropic response is more pronounced as the disease becomes more advanced. Moreover, in chronic heart failure myocardial G_i-protein, which inhibits cAMP formation, is increased, which might further contribute to the reduction in β-adrenoceptor-mediated effects. It appears that, at present, the best therapy for severe heart failure is a successful heart transplant, since in the transplanted heart β-adrenoceptor number and function seems to be noramalized. Moreover, the data currently available do not suggest any development of super- or subsensitivity of postsynaptic cardiac β-adrenoceptors in the transplanted human heart.     

Glutaminyl cyclase activity is a characteristic feature of human cerebrospinal fluid

BACKGROUND: Proteins and peptides occurring in human body fluids can be useful biological markers for neurological diseases and can even contribute to the pathogenesis of such diseases. However, proteins and peptides are potential substrates of proteases and other enzymes. Proteolysis and enzymatic modification may lead to their degradation and modification. METHODS: Using mass spectrometry we investigated the degradation and modification of indicator peptides in the presence of cerebrospinal fluid (CSF). We further applied a fluorometric assay to study the activity of the presumed enzyme glutaminyl cyclase. RESULTS: In CSF we observed an aminopeptidase activity that could partially be inhibited by protease inhibitors and EDTA. In addition, the formation of pyroglutamate (pGlu) from N-terminal glutamine (Gln) was regularly observed. The reaction to pGlu was rapid and protected the indicator peptides from further N-terminal degradation. The conversion of Gln to pGlu could be attributed to the activity of the enzyme glutaminyl cyclase (QC). The QC activity was a characteristic feature of all 45 CSF samples collected from multiple sclerosis patients and controls. CONCLUSION: Glutaminyl cyclase activity is a characteristic feature of human cerebrospinal fluid. The presence of QC in CSF can stabilize peptides from degradation by aminopeptidases. This may have impact for neurological disorders that are characterized by both, the presence of QC and the occurrence of appropriate peptide substrates.     

Two-dimensional gel electrophoresis, isoelectric focusing and agarose gel electrophoresis in the diagnosis of multiple sclerosis

Two dimensional gel electrophoresis (2-DE), isoelectric focusing (IEF) and agarose gel electrophoresis (AGE) were used to examine cerebrospinal fluid (CSF) and sera from 22 patients with confirmed multiple sclerosis, 11 patients with probable multiple sclerosis and 20 control patients with non-inflammatory neurological diseases of the central nervous system (CNS). All of the 22 patients with confirmed multiple sclerosis showed abnormal patterns of oligoclonal IgG in all three methods. In the CSF from patients with probable multiple sclerosis, oligoclonal IgG was detectable in 18 percent with AGE, in 72 percent with IEF and 90 percent with 2-DE. No oligoclonal IgG was observed in subjects with non-inflammatory neurological diseases. Many artefacts in IEF, which lead to misinterpretation, are eliminated in the 2-DE system. Based on our observations and this study in particular, it is evident that some patients have IgG changes which can be detected only by 2-DE. The application of research-oriented 2-DE for routine clinical purposes is still limited by its cost and technical complexity.     

Increase in plasma concentration of ubiquitin in dialysis patients: Possible involvement in β2-microglobulin amyloidosis

β₂-Microglobulin(β2-M) amyloidosis is a serious complication of dialysis therapy; however, its pathogenesis is still unclear. Recent studies have indicated that ubiquitin has amyloid enhancing factor activity, raising the possibility that ubiquitin plays a role in β2-M amyloidogenesis. In this study, synovial tissue from patients with long-term dialysis was examined immunohistochemically. The synovial tissue was labeled with anti-ubiquitin antibody, indicating co-deposition of ubiquitin with β2-M amyloid. To elucidate the involvement of plasma ubiquitin, we established a specific radioimmunoassay for ubiquitin. Using this method, we observed that the plasma concentration of ubiquitin-like immunoreactivity in dialysis patients was significantly higher than that in normal subjects. In chronic renal failure patients, the plasma concentration of ubiquitin-like immunoreactivity was also significantly higher than that in normal controls, which finding suggests that a reduction in renal clearance is, at least in part, responsible for the increased plasma concentration of ubiquitin. In dialysis patients, plasma concentrations of ubiquitin-like immunoreactivity in patients with carpal tunnel syndrome, one of the major symptoms of β2-M amyloidosis, were significantly higher than those in patients not exhibiting this syndrome. These results suggest a possible involvement of plasma ubiquitin in β2-M amyloidosis.     

Peripheral inflammation modifies the effect of intrathecal IL-1beta on spinal PGE2 production mainly through cyclooxygenase-2 activity. A spinal microdialysis study in freely moving rats

Acute inflammation induces upregulation of IL-1β both at the site of the peripheral inflammation and in the cerebrospinal fluid (CSF). The central increase of IL-1β mainly contributes to the development of hypersensitivity. However, the spinal mechanisms for the effects of IL-1β in nociceptive transmission are incompletely understood. It is also unknown whether previous sensitization changes IL-1β activity. We therefore investigated the dose–effect relationship of intrathecal (i.t.) IL-1β on spinal PGE₂ production in the absence and presence of peripheral formalin inflammation with spinal microdialysis in freely moving rats. The possible involvement of cyclooxygenase (COX) isoforms in the IL-1β-mediated spinal PGE₂ production on the background of peripheral formalin inflammation was further evaluated with the selective COX-1 and COX-2 inhibitors. We found that the i.t. administration of IL-1β, with doses of 1, 2, 8, or 16 ng, increased PGE₂ levels in CSF in a dose-related fashion. This IL-1β-evoked PGE₂ release occurred within 30 min after IL-1β administration, peaked at 30–60 min interval, and returned gradually to the baseline level within 4 h. Peripheral formalin inflammation in the paw induced a more prolonged effect of spinal IL-1β with larger PGE₂ releases in the CSF compared with the non-inflammatory state, suggesting that peripheral inflammation enhances central sensitization. The COX-2 inhibitor SC58236 (15 mg/kg) reduced the IL-1β-mediated PGE₂ increase in CSF by 86% while the COX-1 inhibitor SC58560 (15 mg/kg) had less effect (28%). Our study suggests that mainly the COX-2 enzyme mediates the IL-1β-induced increase in spinal PGE₂ in the presence of peripheral formalin inflammation.     

Children with multiple erythema migrans: are there any pre-treatment symptoms and/or signs suggestive for central nervous system involvement?

OBJECTIVE: To establish eventual signs and symptoms suggestive for central nervous system involvement in children with multiple erythema migrans. METHODS: Patients younger than 15 years with multiple erythema migrans, diagnosed at our department from 1996 to 2000, were included in this prospective study. Demographic, clinical and laboratory findings were obtained and compared for a group of patients with pleocytosis (interpreted as a sign of central nervous system involvement) and a group of children with normal cerebrospinal fluid findings. RESULTS: Cerebrospinal fluid pleocytosis (cell counts > or = 5 x 10(6)/l) was detected in 55/214 (25.7%) children; it was lymphocytic in 94.5% of patients and ranged from 5 to 320 (median, 10 x 10(6)/l). Compared with the group with normal cerebrospinal fluid findings, patients with pleocytosis more often reported having had Lyme borreliosis in the past (8/55 versus 3/159; p = 0.0011), had longer incubation periods (25.5 versus 14 days; p = 0.0269), larger diameter of the largest erythema migrans at the time of first examination (10 versus 5.5 cm; p = 0.0055), higher frequency of associated systemic symptoms (45.5% versus 21.4%; p = 0.0011), positive meningeal signs (10.9% versus 1.9%; p = 0.0100), borrelial IgG antibodies in cerebrospinal fluid (3/49 versus 0/150; p = 0.0142) and B. burgdorferi s.l. isolated from cerebrospinal fluid (7/52 versus 1/147; p = 0.0004), but less often had mild initial disease (67.3% versus 88.7%; p = 0.0006). CONCLUSIONS: Cerebrospinal fluid pleocytosis was detected in 25.7% of children with multiple erythema migrans. Although several clinical and laboratory abnormalities were present significantly more often in patients with elevated cell counts than with normal cerebrospinal fluid findings, discriminatory significance for the majority of these abnormalities was low, particularly because of low negative predictive values. In more than 2/3 of patients with pleocytosis the initial disease was mild, fewer than 1/2 reported systemic symptoms, and meningeal signs were expressed in only 11%.