Homocarnosine in human cerebrospinal fluid: an age-dependent phenomenon

—Homocarnosine, rather than carnosine, is the major imidazole dipeptide present in human CSF. This compound can be readily detected in the CSF of normal infants and young children, while it is either not detectable or is present only in very small concentrations in the CSF of normal adult subjects. Slightly greater amounts of homocarnosine can be found in the CSF of some adults with neurological disorders.     

The origin of CSF choline and its relation to acetylcholine metabolism in brain.

Elevation of serum choline levels in the rabbit either by intravenous injection of choline or by the pharmacological action of oxotremorine results in a rise in cisternal CSF choline levels. It was excluded that the oxotremorine induced rise in CSF choline levels can be ascribed to its action on the CNS. Therefore changes in CSF choline levels can be merely the result of changes in peripheral choline stores and do not necessarily reflect changes in the cholinergic activity of the CNS. From isotopic labelling experiments the contribution of serum choline to CSF choline was found to be 42%.     

Cerebellomedullary cerebrospinal fluid collection in the dog

Analysis of cerebrospinal fluid (CSF) can be a valuable diagnostic tool. This column describes cerebellomedullary CSF collection in the dog.     

Correlation between culture of Mycobacterium tuberculosis and antimycobacterial antibody in lumbar, ventricular and cisternal cerebrospinal fluids of patients with tuberculous meningitis.

In this study positive culture for M. tuberculosis were obtained, 20% in lumbar cerebrospinal fluid (CSF), 75% in ventricular CSF and 87.5% in cisternal CSFs of patients with tuberculous meningitis. Low culture positivity in lumbar CSF is due to the low density of circulating tubercle bacilli in lumbar CSF than in cisternal or ventricular CSFs. However antimycobacterial antibody in lumbar, cisternal and ventricular CSFs circulate in significant titres and are not statistically different from one another. Since specimens of CSF can not be obtained from cisternal or ventricular routes for the routine bacteriological investigations in patients with tuberculous meningitis, detection of antimycobacterial antibody of M. tuberculosis antigen 5 in lumbar CSF by an indirect ELISA may be considered as an aid for the diagnosis of tuberculous meningitis, particularly when repeated CSF cultures are negative for M. tuberculosis.     

The impact of blood contamination on the proteome of cerebrospinal fluid

Human cerebrospinal fluid (CSF) is in direct contact with the brain extracellular space. Beside the secretion of CSF by the choroid plexus the fluid also derives directly from the brain by the ependymal lining of the ventricular system and the glial membrane and from blood vessels in the arachnoid. Therefore, biochemical change in the brain may be reflected in the CSF. CSF is a potential source of protein molecular indices of central nervous system function and pathology. However, various amounts of blood contamination in CSF may arise during sample acquisition. The concentration of protein in the CSF is only 0.2 to 0.5% that of blood. Minor contamination of CSF with blood during collection of the fluid may dramatically alter the protein profile confounding the identification of potential biomarkers. We have analyzed CSF and CSF spiked with increasing amounts of whole blood using proteomic techniques. We detected at least four blood specific highly abundant proteins: hemoglobin, catalase, peroxiredoxin and carbonic anhydrase I. These proteins can be used as blood contamination markers for proteomic analysis of CSF. Proteins in blood contaminated CSF samples were less stable compared to neat CSF at 37 degrees C suggesting that blood borne protease may induce protein degradation in CSF during sample acquisition. This analysis was aimed at identification of proteins found primarily in CSF, those found primarily in blood and assessment of the impact of blood contamination on those proteins found in both fluids.     

CSF-studies in neuropsychiatric disorders

Cerebrospinal fluid (CSF) is a clear and colourless fluid that surrounds the brain and spine. Due to the close proximity of CSF to the brain, pathological brain-processes are likely to be reflected in CSF. CSF can be obtained through lumbar puncture and is frequently performed in the differential diagnosis of neuropsychiatric disorders. Beyond clinical applications, CSF has been studied as part of different research-protocols. In this review, we will focus on CSF-analysis in Alzheimer Disease, major depression and schizophrenia. We will review both clinical applications as well as research applications in all three disorders. We will also assess new technological advances that have made it possible to study large numbers of proteins in CSF and how these advances may change CSF-analysis in the years to come.     

Biochemical enrichment of lymphokines secreted by a cloned helper T lymphocyte

The Mls-reactive murine helper T cell clone L2 produces at least 10 lymphokine activities affecting at least five distinct target cells. Culture conditions can be optimized to enable production of high levels of colony-stimulating factors (CSFs) by lectin stimulation of L2 cells under serum-free conditions. Selective enrichment of three lymphokine activities--interleukin 2, CSF, and interferon--can be achieved by using a combination of phenyl-Sepharose and hydroxyapatite chromatography. At least two types of CSF can be separated by concanavalin A-Sepharose chromatography. The CSF that does not bind to concanavalin A-Sepharose can be enriched to a specific activity of at least 5 X 10(8) U/mg of protein by using ion exchange high-pressure liquid chromatography.     

Predictors of bacterial meningitis in resource-limited contexts: an Angolan case

Background

Despite the great morbidity and mortality that childhood bacterial meningitis (BM) is experiencing in Africa, diagnosis of BM in resource-limited contexts is still a challenge. Several algorithms and clinical predictors have been proposed to help physicians in decision-making but a lot of these markers used variables that are calculable only in well-equipped laboratories. Predictors or algorithm based on parameters that can be easily performed in basic laboratories can help significantly in BM diagnosis, even in resource-limited settings, rural hospitals or health centers.

Results

This retrospective study examined 145 cerebral-spinal fluid (CSF) specimens from children from 2 months to 14 years. CSF specimens were divided into two groups, according to the presence or not of a clinical diagnosis of BM. For each specimen, CSF aspect, CSF white blood cells (WBC) count, CSF glucose and protein concentration were analyzed and statistical analysis were performed. CSF WBC count ≥10/µl is no more a valuable predictor of BM. CSF protein concentration ≥50 mg/dl has a better sensitivity for BM diagnosis and when used with CSF glucose concentration ≤40 mg/dl, can help to diagnose correctly almost all the BM cases. An algorithm including CSF protein concentration, glucose concentration and WBC count has been proposed to rule out BM and to correctly diagnose it.

Conclusions

In resource-limited health centers, the availability of a combination of easy-to-obtain parameters can significantly help physicians in BM diagnosis. The prompt identification of a BM case can be rapid treated or transferred to adequate structures and can modify the outcome in the patient.     

高活性重组hG-CSF的制备

我们构建了新的硫氧还蛋白（Ｔｈｉｏｒｅｄｏｘｉｎ）融合表达载体ｐＥＴＴｒｘＬ和ｐＥＴＴｒｘ－ＨｉｓＬ,它们可使功能蛋白在大肠杆菌胞质中以可溶性形式高效表达。利用此表达系统成功地获得的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白的高效可溶性表达,表达水平达总细胞可溶蛋白的４１％以上。所表达的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白可通过Ｃｕ２＋－ＩＤＡＳｅｐｈａｒｏｓｅＦＦ固相金属螯合层析柱,方便地从细胞破碎可溶上清中直接纯化。所获得的融合蛋白具有ｈＧ－ＣＳＦ特异的生物活性,其比活性达到０．５－１．３３×１０７ｕ／ｍｇ融合蛋白。这样表达的ｈＧ－ＣＳＦ融合蛋白能被ＩｇＡ蛋白酶特异地切割,将ｈＧ－ＣＳＦ从融合蛋白上切下获得与天然蛋白一级结构完全一致的重组ｈＧ－ＣＳＦ 。     

Therapeutic effects on experimental metastatic tumor-bearing mice by vaccination with GM-CSF gene-modified and tumor antigen-pulsed macrophages

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Resistance to outflow of cerebrospinal fluid after central infusions of angiotensin.

Infusions of artificial cerebrospinal fluid (CSF) into the cerebroventricles of conscious rats can raise CSF pressure (CSFp). This response can be modified by some neuropeptides. One of these, angiotensin, facilitates the rise in CSFp. We measured CSFp in conscious rats with a computerized system and evaluated resistance to CSF outflow during infusion of artificial CSF, with or without angiotensin, from the decay kinetics of superimposed bolus injections. Angiotensin (10 ng/min) raised CSFp (P less than 0.05) compared with solvent, but the resistance to CSF outflow of the two groups was similar (P greater than 0.05). Because CSFp was increased by angiotensin without an increase in the outflow resistance, a change in some volume compartment is likely. Angiotensin may raise CSFp by increasing CSF synthesis; this possibility is supported, since the choroid plexuses contain an intrinsic isorenin-angiotensin system. Alternatively, angiotensin may dilate pial arteries, leading to an increased intracranial blood volume.     

Antibody buffering in the brain

The efficacy of chemotherapy on brain tumors is often hindered by the presence of the blood brain barrier. This barrier keeps many systemically administered substances from entering the cerebrospinal fluid (CSF), while allowing intrathecally administered drugs free passage out of that compartment. Therefore, achieving a therapeutic concentration of a cell cycle inhibitor in the CSF for a time long enough to have a cytotoxic effect on slow-growing tumor cells has proven difficult. The ability of an antibody to prolong ligand half-life and bioactivity has been previously described occurring in the plasma. This phenomenon has not yet been described or exploited for use in the CSF compartment. Antibodies often have a longer residence time in the CSF than small-molecule drugs, so antibody buffering, administration of a drug with its specific antibody, can prolong the bioactive lifetime of a drug in the CSF. Here we describe antibody buffering of the small molecule hapten 2-phenyl-oxazol-5-one-methylene-gamma-amino butyrate in the CSF of a rats. Not only does the presence of an antibody buffer increase the half-life of both total and free hapten in the CSF, but the antibody can be re-charged in situ with fresh hapten, even days after the initial antibody infusion. Antibody buffering may provide a viable option for delivering a stable, bio-available concentration of a drug that is normally rapidly eliminated from the CSF.     

The presence of cocaine and benzoylecgonine in rat cerebrospinal fluid after the intravenous administration of cocaine.

Cocaine hydrochloride, in doses of 0.5, 1.0, 2.0 and 4.0 mg/kg, iv, was administered to male Sprague-Dawley rats. Cerebrospinal fluid (CSF) was collected from the cisterna magna over a 20 min period and blood samples were obtained at 20 min after cocaine administration. In addition, blood samples for the 1 mg/kg dose of cocaine were collected at 2, 10, 20 and 30 min following drug injection. Gas chromatography/mass spectrometry was used for the analysis of cocaine and its metabolites in plasma and CSF. The disappearance of cocaine (1 mg/kg) from the plasma exhibited first order kinetics with a half-life of 18.11 +/- 3.22 min. Cocaine and benzoylecgonine were found in CSF and the concentrations of cocaine and benzoylecgonine increased in CSF as the doses of cocaine were increased. CSF flow rates were not altered by the iv administration of cocaine or benzoylecgonine. The CSF-to-plasma ratios for cocaine were quite similar to each other over the dosage range of cocaine that was administered; however, the CSF-to-plasma ratios for benzoylecgonine decreased as the concentrations of benzoylecgonine increased in plasma and CSF. When benzoylecgonine (2 mg/kg, iv) was given, the compound was detected in CSF indicating that benzoylecgonine can enter into the central nervous system from the peripheral blood. This investigation shows that cocaine and benzoylecgonine can be assayed in CSF and that the plasma levels of these compounds correlate with their concentrations in CSF.     

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)}     

A Multi-Target Real-Time PCR Assay for Rapid Identification of Meningitis-Associated Microorganisms

A central nervous system (CNS) infection, such as meningitis, is a serious and life-threatening condition. Bacterial meningitis can be severe and may result in brain damage, disability or even death. Rapid diagnosis of CNS infections and identification of the pathogenic microorganisms are needed to improve the patient outcome. Bacterial culture of a patient??s cerebrospinal fluid (CSF) is currently considered the ??gold standard?? for diagnosing bacterial meningitis. From the CSF cultures researchers can assess the in vitro susceptibility of the causative microorganism to determine the best antibiotic treatment. However, many of the culture assays, such as microscopy and the latex agglutination test are not sensitive. To enhance pathogen detection in CSF samples we developed a multi-target real-time PCR assay that can rapidly identify six different microorganisms: Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, Streptococcus agalactiae, Listeria monocytogenes and Cryptococcus neoformans. In this study we applied this PCR analysis to 296 CSF samples from patients who were suspected of having meningitis. Of the 296 samples that were examined, 59 samples were positive according to the CSF culture and/or molecular assays. Forty-six CSF samples were positive for both the CSF culture and our real-time PCR assay, while 13 samples were positive for the real-time PCR but negative for the traditional assays. This discrepancy may have been caused by the fact that these samples were collected from 23 patients who were treated with antimicrobials before CSF sampling.     

Piglets produced from cloned blastocysts cultured in vitro with GM‐CSF

In general, pig embryos established by somatic cell nuclear transfer (SCNT) are transferred at the one‐cell stage because of suboptimal embryo culture conditions. Improvements in embryo culture can increase the practical application of late embryo transfer. The goal of this study was to evaluate embryos cultured with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in vitro, and to track the in vivo developmental competency of SCNT‐derived blastocysts from these GM‐CSF embryos. The receptor for GM‐CSF was up‐regulated in in vitro‐produced embryos when compared to in vivo‐produced cohorts, but the level decreased when GM‐CSF was present. In vitro fertilized (IVF) embryos, supplemented with GM‐CSF (2 or 10 ng/ml), showed a higher frequency of development to the blastocyst stage compared to controls. The total cell numbers of the blastocysts also increased with supplementation of GM‐CSF. Molecular analysis demonstrates that IVF‐derived blastocysts cultured with GM‐CSF exhibit less apoptotic activity. Similarly, an increase in development to the blastocyst stage and an increase in the average total‐cell number in the blastocysts were observed when SCNT‐derived embryos were cultured with either concentration of GM‐CSF (2 or 10 ng/ml). When SCNT‐derived embryos, cultured with 10 ng/ml GM‐CSF, were transferred into six surrogates at Day 6, five of the surrogates became pregnant and delivered healthy piglets. Our findings suggest that supplementation of GM‐CSF can provide better culture conditions for IVF‐ and SCNT‐derived embryos, and pig SCNT‐derived embryos cultured with GM‐CSF in vitro can successfully produce piglets when transferred into surrogates at the blastocyst stage. Thus, it may be practical to begin performing SCNT‐derived embryo transfer at the blastocyst stage. Mol. Reprod. Dev. © 2013 Wiley Periodicals, Inc.     

Biotransformation of nitric oxide in the cerebrospinal fluid of amyotrophic lateral sclerosis patients

Recent findings indicate that nitric oxide (NO*) over-production might be an important factor in the pathogenesis of sporadic amyotrophic lateral sclerosis (SALS). We measured significantly higher concentrations of uric acid and thiol group-containing molecules (R-SH groups) in the cerebrospinal fluid (CSF) from SALS patients compared to controls. The above factors, together with a slightly increased free iron concentration found in the CSF, favour conditions necessary for the formation of the dinitrosyl iron complex, capable of NO* bio-transformation. Thus, we performed ex vivo saturation of CSF (from both SALS patients and controls) with NO*. A decrease in the level of R-SH was found. This was more pronounced in the CSF from SALS patients. In the CSF from SALS patients the production of nitrite and hydroxylamine was greater than that observed in the CSF from controls. Moreover, we also found increased Cu,Zn-SOD activity in the CSF from SALS patients (when compared to control subjects) but no activity corresponding to Mn-SOD in any CSF samples. As Cu,Zn-SOD can react with nitroxyl forming NO*, the conditions for a closed, but continuous, loop of NO* biotransformation are present in the CSF of ALS patients.     

Leptomeningeal carcinomatosis presenting as eosinophilic meningitis

The case of a 67-year-old man with underlying carcinomatous meningitis who presented with meningismus and cerebrospinal fluid (CSF) eosinophilia is reported. CSF eosinophilia can reflect a number of underlying conditions; however, carcinomatous meningitis is not generally considered. In this case, studies for bacterial, fungal and parasitic agents were negative. Cytologic examination of a lumbar puncture specimen revealed malignant epithelial cells in an inflammatory background. When unexplained eosinophilia is found in the CSF, a thorough search for coincident meningeal carcinomatosis should be undertaken.     

Release from mouse macrophages of acidic isoferritins that suppress hematopoietic progenitor cells is induced by purified L cell colony stimulating factor and suppressed by human lactoferrin

Purified mouse L cell colony-stimulating factor (CSF) and purified iron-saturated human lactoferrin (LF) were assessed for their effects on release of acidic isoferritin-inhibitory activity (AIFIA) from resident peritoneal and spleen macrophages of B6D2F1 mice. Constitutive release of AIFIA was dependent on the number of macrophages conditioning the culture medium. Detection of release of AIFIA required at least 10(4) macrophages/ml, and increased release was noted with increased concentrations of cells. This release was enhanced by CSF and was induced by CSF from concentrations of 10(3) macrophages/ml, from which constitutive release of AIFIA was not detected. Increased concentrations of CSF induced increased release of AIFIA. The inducing effect was removed by pretreating CSF with rabbit anti-L cell CSF serum. LF suppressed the constitutive as well as the CSF-induced release of AIFIA, but results were dependent on the relative concentrations of LF and CSF used. The suppressive effects of LF were removed by pretreating LF with goat anti-human LF. Constitutive, but not CSF-induced, release of AIFIA could be ablated by removal of Ia antigen-positive macrophages with low concentrations of monoclonal anti-Ia plus complement. Treating macrophages with higher concentrations of anti-Ia in the absence of complement blocked the LF suppression of constitutive AIFIA release but not the CSF-induction of AIFIA release. Release of AIFIA from mouse macrophages can be modulated by CSF and LF. This modulation may be of significance for the regulation of myelopoiesis.