A More Sensitive Radioimmunoassay for Neuron-Specific Enolase Suitable for Cerebrospinal Fluid Determinations

Abstract: Neuron-specific enolase (NSE) and non-neuronal enolase (NNE) have been shown to be highly specific neuronal and glial products respectively and are therefore useful as biochemical markers of the two major cell types in the vertebrate central nervous system. An iodinated radioimmunoassay (RIA) procedure for human NSE (NSE-H) with approximately 50-fold greater sensitivity than the previously available tritiated assay is described. This assay is capable of detecting 100 pg of NSE-H per assay. NSE levels in human cerebrospinal fluid (CSF) which were previously undetectable with the tritiated RIA are now easily measured and have been shown to be approximately 2 ng/ml of CSF. Furthermore, results obtained with the newly described assay procedure on more concentrated brain tissue extracts are comparable to the tritiated RIA. The iodinated NSE RIA is also shown to be capable of accurately detecting added amounts of NSE in human CSF, indicating the potential clinical usefulness of this assay in determining elevated levels of NSE in CSF.     

Characterization of Nitrotyrosine-Modified Proteins in Cerebrospinal Fluid

Background

HIV-associated neurocognitive disorders (HAND) has been associated with the up-regulation of various oxidative stress pathways. Previous studies have linked the neuronal damage observed in individuals diagnosed with HAND to increased nitrotyrosine modification of neuronal proteins.

Materials and methods

Tyrosine nitration alters protein structure and function, affects biological half-life, and potentially prevents the phosphorylation of key tyrosine residues involved in signal transduction pathways. Therefore, in this study we employed proteomics-based experimental approaches to investigate nitrotyrosine-modified proteins in pooled cerebrospinal fluid (CSF) of individuals diagnosed with HAND. To identify specific nitrotyrosine-modified proteins in the CSF of individuals diagnosed with HAND, affinity purification and high-performance tandem mass spectrometry are utilized in a “bottom-up” proteomics approach.

Results

From tandem mass spectrometric analysis, we identified major proteins that underwent nitration as a result of nitro-oxidative stress in the CSF of individuals diagnosed with HAND. We also utilized analytical and biochemical techniques to characterize the expression and modification site of in vivo nitrated lipocalin-type prostaglandin-D synthase in HAND CSF.     

Thinprep plus Papanicolaou Stain Method Is More Sensitive than Cytospin-Coupled Wright Giems Stain Method in Cerebrospinal Fluid Cytology for Diagnosis of Leptomeningeal Metastasis from Solid Tumors

Background

The present study was designed to determine whether the Thinprep plus Papanicolaou stain (Thinprep) method is more sensitive than the Cytospin-coupled Wright-Giemsa (WG) stain (Cytospin) method in diagnosis of leptomeningeal metastasis (LM) from malignant solid tumors in cerebrospinal fluid (CSF). We also explored if the Thinprep method could be used in the differential diagnosis of the type of primary tumor cells based on the morphology of tumor cells in CSF samples.

Methods

The morphological features of tumor cells in fresh CSF samples were analyzed using both methods. The tumor cell detection rates were compared between the two methods.

Results

Using the Thinprep method, we found that each type of tumor cells in the CSF samples had specific identifiable morphological features linked to their primary cancer origins, such as adenocarcinomas originated from the lungs, breast, and stomach, and lung squamous cell carcinomas, small cell lung cancer, large-cell neuroendocrine lung cancer, hepatocellular carcinoma, and malignant melanoma. In a retrospective study with 88 LM patients, cancer cells were detected in 80 out of the 88 CSF samples. In the comparative study with 45 LM patients, the initial detection rate of the Thinprep method was significantly higher than that of the Cytospin method (73.3% vs. 57.8%, P<0.01). The cell morphology was better preserved and subcellular structures were clearer using the Thinprep method, compared to the Cytospin method.

Conclusions

The Thinprep method is more sensitive and suitable for LM diagnosis in CSF in patients with malignant solid tumors than the Cytospin method. The Thinprep method may facilitate primary tumor detection and help design early treatment regimens for LM patients with tumors of unknown primary origin.     

Amine Metabolites in the Lumbar Cerebrospinal Fluid of Humans with Restricted Flow of Cerebrospinal Fluid

DETERMINATION of homovanillic acid (HVA) and 5-hydroxy-indole acetic acid (5HIAA) in human lumbar cerebrospinal fluid (CSF) is becoming an important tool in the study of the metabolism in the brain of their respective precursors, dopamine and 5-hydroxytryptamine and in the interpretation of the effects of drugs on these substances. The assumption that the concentration of the acidic metabolites HVA and 5HIAA in the lumbar CSF gives a measure of the amount of turnover of the parent amines in the brain is supported by several findings. (1) Amine metabolite concentrations in the lateral ventricular CSF of the dog correlate with their concentrations in adjacent brain areas¹. (2) Peripherally administered HVA only penetrates slightly or not at all to lateral ventricular CSF in the cat² or dog³, similar results being obtained for 5HIAA in the dog⁴. (3) Drugs which alter brain amine turnover in laboratory animals also alter the concentrations of the acidic metabolites in dog³, rabbit⁵ and human⁶ CSF in the appropriate direction. (4) In Parkinsonism and in senile and presenile dementia, conditions in which there is evidence of defective turnover of amines in the brain, low concentrations of HVA and 5HIAA are found in the CSF⁷.     

Detection of Proteins in Normal and Alzheimer's Disease Cerebrospinal Fluid with a Sensitive Sandwich Enzyme-Linked Immunosorbent Assay

Abstract— Alzheimer's disease is a progressive degenerative dementia characterized by the abundant presence of neurofibrillary tangles in neurons. This study was designed to test whether the microtubule-associated protein, a major component of neurofibrillary tangles, could be detected in CSF. Additionally, we investigated whether CSF levels were abnormal in Alzheimer's disease as compared with a large group of control patients. We developed a sensitive sandwich enzyme-linked immunosorbent assay using AT120, a monoclonal antibody directed to human, as a capturing antibody. With this technique, the detection limit for was less than 5 pg/ml of CSF. Using ATS, which recognizes abnormally phosphorylated ser-ines 199–202 in, the detection limit was below 20 pg/ml of CSF. However, with AT8, we found no immunoreactiv-ity in CSF, suggesting that only a small fraction of CSF contains the abnormally phosphorylated AT8 epitope. Our results indicate that CSF levels are significantly increased in Alzheimer's disease. Also, CSF levels in a large group of patients with a diversity of neurological diseases showed overlap with CSF levels in Alzheimer's disease.     

Immunoglobulin Levels in the Cerebrospinal Fluid

The immunoglobulins G, A, M, and D have been measured in the cerebrospinal fluid of 207 patients with neurological disease. Raised levels of IgG, expressed as a percentage of total cerebrospinal fluid (C.S.F.) protein, were found in 62% of 45 cases of multiple sclerosis compared with 14% of 160 cases with various other neurological disorders. Thus measurement of the IgG level is probably a useful confirmatory investigation in multiple sclerosis. IgA and IgM were found only in the C.S.F. of patients with a raised protein level, and IgD was not detected.     

A Technique for Serial Collection of Cerebrospinal Fluid from the Cisterna Magna in Mouse

Alzheimer''s disease (AD) is a progressive neurodegenerative disease that is pathologically characterized by extracellular deposition of β-amyloid peptide (Aβ) and intraneuronal accumulation of hyperphosphorylated tau protein. Because cerebrospinal fluid (CSF) is in direct contact with the extracellular space of the brain, it provides a reflection of the biochemical changes in the brain in response to pathological processes. CSF from AD patients shows a decrease in the 42 amino-acid form of Aβ (Aβ42), and increases in total tau and hyperphosphorylated tau, though the mechanisms responsible for these changes are still not fully understood. Transgenic (Tg) mouse models of AD provide an excellent opportunity to investigate how and why Aβ or tau levels in CSF change as the disease progresses. Here, we demonstrate a refined cisterna magna puncture technique for CSF sampling from the mouse. This extremely gentle sampling technique allows serial CSF samples to be obtained from the same mouse at 2-3 month intervals which greatly minimizes the confounding effect of between-mouse variability in Aβ or tau levels, making it possible to detect subtle alterations over time. In combination with Aβ and tau ELISA, this technique will be useful for studies designed to investigate the relationship between the levels of CSF Aβ42 and tau, and their metabolism in the brain in AD mouse models. Studies in Tg mice could provide important validation as to the potential of CSF Aβ or tau levels to be used as biological markers for monitoring disease progression, and to monitor the effect of therapeutic interventions. As the mice can be sacrificed and the brains can be examined for biochemical or histological changes, the mechanisms underlying the CSF changes can be better assessed. These data are likely to be informative for interpretation of human AD CSF changes.Open in a separate windowClick here to view.^{(49M, flv)}     

Evidence for Two Cholesterol Ester Hydrolases in Human Cerebrospinal Fluid

Abstract: In the present study, the properties, such as pH optima, detergent requirement, and effects of various lipids, of cholesterol ester hydrolase in human cerebrospinal fluid (CSF) were examined, and the activity levels of the enzyme in CSF from multiple sclerosis (MS) patients and non-MS individuals were compared. Our data indicate that the enzyme in CSF exhibits two pH optima: pH 6.0 in the presence of Triton X-100 and pH 7.0 in the presence of sodium taurocholate. Both phosphatidylethanolamine (PE) and phosphatidylserine (PS) enhanced the hydrolase activity at pH 6.0. The activity at pH 7.0, on the other hand, was enhanced slightly in the presence of PE but was inhibited in the presence of PS. These data suggest the presence of two cholesterol ester hydrolases in CSF and also indicate that the activity at pH 6.0 may be due to microsomal enzyme in brain and that at pH 7.0 may be due to myelin enzyme. The hydrolase activity at pH 7.0 was significantly lower in CSF from MS patients. The activity at pH 6.0 in CSF from MS and non-MS patients, however, did not differ significantly. This indicates that the reduction in pH 7.0 hydrolase activity in CSF may be related to demyelination.