Cerebrospinal Fluid Analysis in Multiple Sclerosis Diagnosis: An Update.

Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system (CNS) with brain neurodegeneration. MS patients present heterogeneous clinical manifestations in which both genetic and environmental factors are involved. The diagnosis is very complex due to the high heterogeneity of the pathophysiology of the disease. The diagnostic criteria have been modified several times over the years. Basically, they include clinical symptoms, presence of typical lesions detected by magnetic resonance imaging (MRI), and laboratory findings. The analysis of cerebrospinal fluid (CSF) allows an evaluation of inflammatory processes circumscribed to the CNS and reflects changes in the immunological pattern due to the progression of the pathology, being fundamental in the diagnosis and monitoring of MS. The detection of the oligoclonal bands (OCBs) in both CSF and serum is recognized as the "gold standard" for laboratory diagnosis of MS, though presents analytical limitations. Indeed, current protocols for OCBs assay are time-consuming and require an operator-dependent interpretation. In recent years, the quantification of free light chain (FLC) in CSF has emerged to assist clinicians in the diagnosis of MS. This article reviews the current knowledge on CSF biomarkers used in the diagnosis of MS, in particular on the validated assays and on the alternative biomarkers of intrathecal synthesis.

Acetylcholinesterase activity in the rat brain after pneumococcal meningitis.

Pneumococcal meningitis is a life-threatening disease characterized by acute purulent infection of the meninges causing neuronal injury, cortical necrosis and hippocampal apoptosis. Cholinergic neurons and their projections are extensively distributed throughout the central nervous system. The aim of this study was to assess acetylcholinesterase activity in the rat brain after pneumococcal meningitis. In the hippocampus, frontal cortex and cerebrospinal fluid, acetylcholinesterase activity was found to be increased at 6, 12, 24, 48 and 96 hr without antibiotic treatment, and at 48 and 96 hr with antibiotic treatment. Our data suggest that acetylcholinesterase activity could be related to neuronal damage induced by pneumococcal meningitis.

Immunocapture-based fluorometric assay for the measurement of neprilysin-specific enzyme activity in brain tissue homogenates and cerebrospinal fluid.

Neprilysin, a zinc-metalloendopeptidase, has important roles in the physiology and pathology of many diseases such as hypertension, cancer and Alzheimer's disease. We have developed an immunocapture assay to measure the specific enzyme activity of neprilysin in brain tissue homogenates and cerebrospinal fluid (CSF). The assay uses a neprilysin-specific antibody, previously used in a commercially available ELISA kit, to isolate and immobilise NEP from brain homogenates and CSF, prior to the addition of a fluorogenic peptide substrate (Mca-RPPGFSAFK(Dnp)). This fluorogenic substrate is ordinarily cleaved by multiple enzymes. We have shown that without the immunocapture phase, even under reaction conditions reported to be specific for neprilysin - i.e. in the presence of thiorphan, at pH above 7 - the fluorogenic peptide substrate does not allow neprilysin activity in brain homogenates and CSF to be discriminated from that of other closely related enzymes. The specificity of the immunocapture enzyme activity assay was confirmed by >80% inhibition of substrate cleavage in brain homogenates and CSF in the presence of thiorphan. The assay allows high-throughput analysis and, critically, also ensures a high level of enzyme specificity even when assaying crude tissue homogenates or CSF.

Matrix metalloprotease-9 in cerebrospinal fluid correlates with disease activity in lymphomatous meningitis.

PURPOSE: The detection of lymphoma cells in cerebrospinal fluid (CSF) as a means to diagnose lymphomatous meningitis is problematic due to its low sensitivity. We hypothesize that matrix metalloproteases (MMPs) would be important in lymphomatous meningitis because lymphoma cells may produce MMPs for brain invasion and angiogenesis. PATIENTS AND METHODS: Twentynine samples of CSF collected longitudinally from 5 patients receiving treatments for primary or metastatic CNS lymphomas were retrospectively analyzed by zymography. Cerebrospinal fluid cytology was correlated with levels of total protein, glucose, MMP-2, MMP-9, activated MMP-9, and 130 kDa MMP. RESULTS: Increased CSF white blood cells (65 +/- 32 cells/microL vs. 9 +/- 8 cells/microL; P = 0.03) and MMP-9 (12.108 +/- 2.675 vs. 9.359 +/- 1.936; P = 0.02) had a strong correlation with abnormal CSF cytology. In addition, we observed that activated MMP-9 would appear and disappear depending on disease activity. Although there was nearly a 4-fold increase of mean activated MMP-9 in CSF samples with abnormal cytology findings when compared with negative cytology findings, the correlation did not reach statistical significance (1.382 +/- 0.76 vs. 0.389 +/- 0.155; P = 0.17). CONCLUSION: Matrix metalloprotease-9 correlated strongly with lymphomatous meningitis, but MMP-2, activated MMP-2, activated MMP-9, and 130-kDa MMP did not.

Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer's Disease Subjects.

Ferroxidase activity has been reported to be altered in various biological fluids in neurodegenerative disease, but the sources contributing to the altered activity are uncertain. Here we assay fractions of serum and cerebrospinal fluid with a newly validated triplex ferroxidase assay. Our data indicate that while ceruloplasmin, a multicopper ferroxidase, is the predominant source of serum activity, activity in CSF predominantly derives from a <10 kDa component, specifically from polyanions such as citrate and phosphate. We confirm that in human biological samples, ceruloplasmin activity in serum is decreased in Alzheimer's disease, but in CSF a reduction of activity in Alzheimer's disease originates from the polyanion component.

Temporal analysis of blood-brain barrier disruption and cerebrospinal fluid matrix metalloproteinases in rhesus monkeys subjected to transient ischemic stroke.

Blood-brain barrier (BBB) disruption plays an important role in pathophysiological progress of ischemic stroke. However, our knowledge of the dynamic change of BBB permeability and its mechanism remains limited. In the current study, we used a non-human primate (NHP) MCAO model and a serial CSF sampling method that allowed us to determine the dynamic change of BBB permeability by calculating the CSF/serum albumin ratio (AR). We showed that AR increased rapidly and significantly after ischemia, and the fold increase of AR is highly correlated with the infarction size during the subacute phase. Moreover, we determined the temporal change of MMP-1, MMP-2, MMP-3, MMP-9, MMP-10, MMP-13, TIMP-1, and TIMP-2 in CSF and serum. Each MMP and TIMP showed different change patterns when comparing their values in CSF and serum. Based on the longitudinal dataset, we showed that the fold increase of MMP-9 in serum and CSF are both correlated to infarction size. Among the measured MMPs and TIMPs, only MMP-2, MMP-13, and TIMP-2 in CSF correlated with AR to some extent. Our data suggest there is no single MMP or TIMP fully responsible for BBB breakdown, which is regulated by a much more complicated signal network and further investigations of the mechanisms are needed.

The V-ATPase is expressed in the choroid plexus and mediates cAMP-induced intracellular pH alterations.

The cerebrospinal fluid (CSF) pH influences brain interstitial pH and, therefore, brain function. We hypothesized that the choroid plexus epithelium (CPE) expresses the vacuolar H+-ATPase (V-ATPase) as an acid extrusion mechanism in the luminal membrane to counteract detrimental elevations in CSF pH. The expression of mRNA corresponding to several V-ATPase subunits was demonstrated by RT-PCR analysis of CPE cells (CPECs) isolated by fluorescence-activated cell sorting. Immunofluorescence and electron microscopy localized the V-ATPase primarily in intracellular vesicles with only a minor fraction in the luminal microvillus area. The vesicles did not translocate to the luminal membrane in two in vivo models of hypocapnia-induced alkalosis. The Na+-independent intracellular pH (pHi) recovery from acidification was studied in freshly isolated clusters of CPECs. At extracellular pH (pHo) 7.4, the cells failed to display significant concanamycin A-sensitive pHi recovery (i.e., V-ATPase activity). The recovery rate in the absence of Na+ amounted to <10% of the pHi recovery rate observed in the presence of Na+ Recovery of pHi was faster at pHo 7.8 and was abolished at pHo 7.0. The concanamycin A-sensitive pHi recovery was stimulated by cAMP at pH 7.4 in vitro, but intraventricular infusion of the membrane-permeant cAMP analog 8-CPT-cAMP did not result in trafficking of the V-ATPase. In conclusion, we find evidence for the expression of a minor fraction of V-ATPase in the luminal membrane of CPECs. This fraction does not contribute to enhanced acid extrusion at high extracellular pH, but seems to be activated by cAMP in a trafficking-independent manner.

Intraperitoneal administration of 340 kDa human plasma butyrylcholinesterase increases the level of the enzyme in the cerebrospinal fluid of rats.

Human butyrylcholinesterase (BuChE) is being developed as a new therapeutic for protection against the toxicity of organophosphorus agents and cocaine. The purified BuChE consists predominantly of 340 kDa tetramers and contains less than 5% monomers and dimers. Our goal was to determine whether BuChE crosses the blood-cerebrospinal fluid (CSF) barrier. Rats were injected intraperitoneally with 1mg of purified human BuChE. Plasma BuChE activity increased nearly 400-fold, while BuChE activity in the CSF increased three-fold. Sucrose density centrifugation showed that the human BuChE molecule in the rat CSF was a tetramer. Immunoprecipitation confirmed the identity of the CSF BuChE as human BuChE. The lower amount of human BuChE in the CSF (0.04%) than of smaller proteins (0.1-1%), with respect to their levels in plasma, supports the idea that passage through the blood-CSF barrier depends on molecular size. BuChE in the CSF could serve to protect the brain from the neurotoxicity of organophosphorus pesticides and cocaine.

Detection of the human telomerase RNA component by in situ hybridization in cells from body fluids.

OBJECTIVE: To apply in situ hybridization (ISH) detection of human telomerase reverse transcriptase (hTERT) mRNA expression in abnormal cells in body fluids to evaluate its usefulness in the diagnosis of malignant effusions. STUDY DESIGN: We investigated the expression of hTERT mRNA by ISH in 33 fluid samples from 30 patients, including 1 cerebrospinal fluid, 18 pleural fluids, 1 pericardial fluid, 12 peritoneal fluids and 1 bronchial washing. Comparison of the results of ISH with those of conventional cytologic evaluation was also performed. RESULTS: Cytomorphologic examination of the 33 body fluids allowed classification as malignant, suspicious, atypical and benign conditions. Among the 17 malignant conditions, there were 15 cases positive by both cytology and ISH. There were 9 discrepant cytology-ISH results in patients with various conditions, including 2 cases positive by cytology and inconclusive by ISH, 5 cases suspicious by cytology and positive by ISH, and 2 cases atypical by cytology and negative by ISH. Among the 9 benign conditions, there were 8 cases negative by both cytology and ISH and 1 case negative by cytology and positive by ISH. This test was highly sensitive (90%) and specific (91 %) and had favorable positive (95%) and negative (83%) predictive values. CONCLUSION: Expression of the hTERT mRNA component can be easily detected by ISH in malignant cells from body fluids. This method is especially useful when the abnormal cellpopulation in the fluid consists of limited numbers of suspicious cells that cannot be completely differentiated from reactive mesothelial cells and thus may help differentiate true positive cases from false negative ones. This ISH method for the detection of expression of the hTERT mRNA component may be an ancillary test for early recognition of cancer cells in body fluids and thus has potential as a diagnostic adjunct in cytopathology.

Gene transfer of extracellular superoxide dismutase increases superoxide dismutase activity in cerebrospinal fluid.

BACKGROUND AND PURPOSE: Copper-zinc superoxide dismutase (CuZnSOD) is expressed intracellularly, while extracellular SOD (EC-SOD) is released from cells. The purpose of this study was to determine whether gene transfer of CuZnSOD increases SOD activity predominantly in tissues, and gene transfer of EC-SOD increases SOD activity in cerebrospinal fluid (CSF). We also determined whether heparin or dextran sulfate releases EC-SOD into CSF. METHODS: We injected recombinant adenoviruses expressing EC-SOD (AdEC-SOD), CuZnSOD (AdCuZnSOD), or beta-galactosidase (Adbeta-gal) into the cisterna magna of rabbits. RESULTS: Total SOD activity in CSF was 39+/-11 U/mL (mean+/-SE) before virus injection. Three days later, total SOD activity in CSF increased to 148+/-22 U/mL after AdEC-SOD and 92+/-10 U/mL after AdCuZnSOD (P:<0.05 versus AdEC-SOD), with no change after Adbeta-gal (49+/-5 U/mL). EC-SOD protein was detected in CSF after AdEC-SOD but not AdCuZnSOD or Adbeta-gal. Injection of heparin or dextran sulfate into the cisterna magna increased total SOD activity 27-fold and 32-fold over basal values, respectively, in CSF of rabbits that received AdEC-SOD. In contrast to effects in CSF, total SOD activity in basilar artery and meninges was significantly higher after AdCuZnSOD and tended to be higher after AdEC-SOD than after Adbeta-gal. CONCLUSIONS: -We have developed a method for intracranial gene transfer of CuZnSOD and EC-SOD. After gene transfer, CuZnSOD was expressed mainly in tissues, and EC-SOD was released into the CSF, especially after injection of heparin or dextran sulfate. Gene transfer of different isoforms of SOD may be useful in studies of cerebral vascular physiology and pathophysiology.

Glutamine synthetase in cerebrospinal fluid, serum, and brain: a diagnostic marker for Alzheimer disease?

OBJECTIVES: To determine whether the glutamine synthetase (GS) level in cerebrospinal fluid (CSF) is a useful biochemical marker in the diagnosis of Alzheimer disease (AD), and to assess the source of GS (brain vs. blood derived) in CSF. METHODS: Sandwich enzyme immunoassay and immunoblotting were applied to detect GS in CSF and in serum from neurologically healthy control subjects and patients with neurodegenerative diseases, including AD. The origin of GS was estimated by the concentration gradients of CSF to serum and ventricular to lumbar CSF. In addition, postmortem brain tissue from controls and patients with AD was analyzed using immunohistochemistry for expression of GS. RESULTS: Levels of GS were significantly increased in lumbar CSF from patients with AD (20+/-12 pg/mL; P = .01) and to a lesser extent in patients with vascular dementia and amyotrophic lateral sclerosis. In CSF of controls, GS levels were 4+/-3 pg/mL. The GS concentration gradients were less than 1:10 for CSF to serum and 2:1 for ventricular to lumbar CSF. Immunoreactivity of GS was most prominent in astrocytes from temporal neocortex of patients with AD, suggesting a relationship between astrocyte reactions and increased GS levels in CSF. CONCLUSIONS: Level of GS in lumbar CSF of patients with AD is increased significantly but nonspecifically, probably related to the strong astrogliosis in brain. Glutamine synthetase in lumbar CSF is mainly brain derived.

Regional study of acid hydrolases and lysosomal membrane properties in the normal human brain at various ages.

Acid hydrolases and lysosomal membrane properties were studied at various ages in the normal human brain. In CSF and four brain regions, the inferior olive, the cerebellar cortex, the caudate nucleus and the frontal cortex were thus beta-galactosidase, beta-glucosidase, alpha-mannosidase, hexosaminidase and acid phosphatase biochemically quantitated at ages varying between 2 and 89 years of age. Also the membrane latency for acid phosphatase was studied in these regions. No major regional quantitative differences were found with regard to the enzymes studied. Their kinetic properties were also defined. There appeared to exist a regional and intra-areal variation in lysosomal membrane permeability. There was, however, no age related increase in total enzyme contents. The possibility significance of these findings are discussed with reference to the aging process.

Decreased glutathione transferase activity in brain and ventricular fluid in Alzheimer's disease.

OBJECTIVE: To investigate the levels of glutathione transferase (GST), a protective enzyme against aldehydes, and especially 4-hydroxynonenal (HNE) in the brain and ventricular CSF of autopsied AD and normal control subjects. BACKGROUND: Studies have implicated increased levels of oxidative stress in the brain in the pathogenesis of AD. Decreased levels of polyunsaturated fatty acids and increased levels of markers of lipid peroxidation have been reported in the brain in AD, particularly in areas severely affected in the disease. HNE, one marker of lipid peroxidation, is neurotoxic in neuronal culture and in vivo and is elevated in AD brain and CSF. METHODS: We measured levels of GST activity and protein in multiple brain regions and ventricular CSF in short-postmortem-interval AD patients and age-matched prospectively evaluated control subjects. RESULTS: A decrease in GST activity in all brain areas was observed in AD compared with controls with significant decreases in the amygdala, hippocampus and parahippocampal gyrus, inferior parietal lobule, and nucleus basalis of Meynert. Levels of GST protein also were depleted in most brain regions in AD. A significant decrease in GST activity and protein levels was also found in ventricular CSF in AD. CONCLUSION: Reduced levels of GST, a protective mechanism against HNE, may have a role in the pathogenesis of neuron degeneration in AD.

Iso-renin of extrarenal origin. "The tissue angiotensinogenase systems".

Enzymes, similar to kidney renin, are present in extrarenal tissue of most mammals; they hydrolyze angiotensinogen to form angiotensin I. We suggest that these enzymes be called angiotensinogenases. Angiotensinogenase concentrations in extrarenal tissue can exceed those in the kidney. The enzyme has been obtained in pure crystalline form. Angiotensinogenases are part of a complex enzyme system which leads to local production of angiotensin. Results indicating a biologic role of the angiotensinogenase system in brain, adrenal gland, uterus and tissue culture are discussed.

Caspase inhibitors protect against neuronal apoptosis induced by cerebrospinal fluid from multiple sclerosis patients.

Neuronal apoptosis has recently been implicated in multiple sclerosis (MS). Apoptotic cell death of neurons is induced in cultures exposed to cerebrospinal fluid (CSF) from MS patients. Since caspases are essential in the regulation of apoptosis, direct evidence was sought linking caspases to CSF-induced neuronal death. Caspase activity was measured in cell extracts from MS CSF-treated cultured neurons by the cleavage of caspase-1 and caspase-3 substrates. Caspase-3 activity, but not caspase-1, was induced in neuronal cultures in response to MS CSF treatment. This caspase-3 activity was inhibited in vitro by Ac-YVAD-cmk and Ac-DEVD-cmk caspase inhibitors. Treatment of MS CSF-incubated neuronal cells with these caspase inhibitors completely preserved neuronal survival and largely attenuated DNA fragmentation detected in situ. These findings show that neuronal cells are rescued from MS CSF-induced death by caspase inhibitors and suggest ways to treat MS.

Matrix metalloproteinase-9 (MMP-9) in human cerebrospinal fluid (CSF): elevated levels are primarily related to CSF cell count.

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<0.0001), but not with CSF/serum albumin ratio (Q(Alb)) (r=0.212; P=0.057), a measure for BBB impairment. Moreover, the CSF/serum MMP-9 ratio (Q(MMP-9)) did not correlate with Q(Alb)(r=0.192; P=0.100). By use of a Boyden chamber, in which granulocytes migrated through a reconstituted basement membrane, it was demonstrated that the MMP-9 concentration in the lower chamber correlated very significantly with the number of accumulated cells (r(2)=0.7692; P<0.0001). The meaning of the increase of MMP-9 in CSF is critically discussed.

Diagnostic significance and source of lactate dehydrogenase and its isoenzymes in cerebrospinal fluid of children with a variety of neurological disorders.

Two hundred and thirty-four cerebrospinal fluid (CSF) specimens from 183 different children were analysed for total lactate dehydrogenase (LD) activity and LD isoenzyme distribution. The LD activities were elevated in the CSF of patients with meningitis, especially with bacterial infections, and with central nervous system (CNS) leukaemia. The CSF LD isoenzyme patterns of both groups generally reflected the number and distribution of lymphocytes and granulocytes in the CSF. Increases in CSF LD levels also occurred in patients with other neurological disorders, such as hydrocephalus, raised intracranial pressure, and epileptic seizures. However, no significant increases in CSF LD activity nor abnormality of the isoenzyme distribution were noted in children who had had a non-specific febrile convulsion.

Acetylcholinesterase assay for cerebrospinal fluid using bupivacaine to inhibit butyrylcholinesterase.

BACKGROUND: Most test systems for acetylcholinesterase activity (E.C.3.1.1.7.) are using toxic inhibitors (BW284c51 and iso-OMPA) to distinguish the enzyme from butyrylcholinesterase (E.C.3.1.1.8.) which occurs simultaneously in the cerebrospinal fluid. Applying Ellman's colorimetric method, we were looking for a non-toxic inhibitor to restrain butyrylcholinesterase activity. Based on results of previous in vitro studies bupivacaine emerged to be a suitable inhibitor. RESULTS: Pharmacokinetic investigations with purified cholinesterases have shown maximum inhibition of butyrylcholinesterase activity and minimal interference with acetylcholinesterase activity at bupivacaine final concentrations between 0.1 and 0.5 mmol/l. Based on detailed analysis of pharmacokinetic data we developed three equations representing enzyme inhibition at bupivacaine concentrations of 0.1, 0.2 and 0.5 mmol/l. These equations allow us to calculate the acetylcholinesterase activity in solutions containing both cholinesterases utilizing the extinction differences measured spectrophotometrically in samples with and without bupivacaine. The accuracy of the bupivacaine-inhibition test could be confirmed by investigations on solutions of both purified cholinesterases and on samples of human cerebrospinal fluid. If butyrylcholinesterase activity has to be assessed simultaneously an independent test using butyrylthiocholine iodide as substrate (final concentration 5 mmol/l) has to be conducted. CONCLUSIONS: The bupivacaine-inhibition test is a reliable method using spectrophotometrical techniques to measure acetylcholinesterase activity in cerebrospinal fluid. It avoids the use of toxic inhibitors for differentiation of acetylcholinesterase from butyrylcholinesterase in fluids containing both enzymes. Our investigations suggest that bupivacaine concentrations of 0.1, 0.2 or 0.5 mmol/l can be applied with the same effect using 1 mmol/l acetylthiocholine iodide as substrate.

Characterization of a brain-related serine protease, neurosin (human kaillikrein 6), in human cerebrospinal fluid.

Neurosin (also known as zyme or protease M) is a trypsin-like serine protease dominantly expressed in the human brain. According to the official nomenclature, this gene is now designated as human kallikrein 6 (KLK6) and the protein is designated hK6. To investigate the metabolism of neurosin in human brain, neurosin contained in the human cerebrospinal fluid (CSF) was analyzed. Neurosin was detected in the all CSFs tested by Western blot analysis using an anti-neurosin monoclonal antibody. We purified neurosin from CSF (CSF-neurosin) using an immunoaffinity chromatography and an anion-exchange chromatography. SDS-PAGE revealed that the purified protein has a relative mol. mass (Mr) of 25,000 Da. The observed sequence of the N-terminal amino acids, Glu-Glu-Gln-Asn-Lys, of the purified CSF-neurosin was identical to the sequence of N-terminal of the pro-enzyme form, which is presumed to have no enzyme activity. CSF-neurosin neither showed any enzyme activity to Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide, which is known to be degraded by the mature neurosin, nor cleaved gelatin. To confirm that the major portion of CSF-neurosin is present in the pro-enzyme form, Western blot analysis using antibodies specific to the pro- or mature enzyme was carried out. The antibody against the mature neurosin fragment did not react with CSF-neurosin. Only the antibody against the pro-enzyme fragment detected CSF-neurosin. Thus, our results suggest that neurosin is present as an inactive pro-enzyme in the human CSF.