Identification of extracellular miRNA in human cerebrospinal fluid by next-generation sequencing

There has been a growing interest in using next-generation sequencing (NGS) to profile extracellular small RNAs from the blood and cerebrospinal fluid (CSF) of patients with neurological diseases, CNS tumors, or traumatic brain injury for biomarker discovery. Small sample volumes and samples with low RNA abundance create challenges for downstream small RNA sequencing assays. Plasma, serum, and CSF contain low amounts of total RNA, of which small RNAs make up a fraction. The purpose of this study was to maximize RNA isolation from RNA-limited samples and apply these methods to profile the miRNA in human CSF by small RNA deep sequencing. We systematically tested RNA isolation efficiency using ten commercially available kits and compared their performance on human plasma samples. We used RiboGreen to quantify total RNA yield and custom TaqMan assays to determine the efficiency of small RNA isolation for each of the kits. We significantly increased the recovery of small RNA by repeating the aqueous extraction during the phenol-chloroform purification in the top performing kits. We subsequently used the methods with the highest small RNA yield to purify RNA from CSF and serum samples from the same individual. We then prepared small RNA sequencing libraries using Illumina’s TruSeq sample preparation kit and sequenced the samples on the HiSeq 2000. Not surprisingly, we found that the miRNA expression profile of CSF is substantially different from that of serum. To our knowledge, this is the first time that the small RNA fraction from CSF has been profiled using next-generation sequencing.     

Identification of neuropeptide FF-related peptides in human cerebrospinal fluid by mass spectrometry

Several neuropeptide FF (NPFF)-related peptides, known as modulators of the opioid system, have been previously characterized in bovine and rodent brain. Reverse-phase high pressure liquid chromatography (HPLC) fractions of a human with normal pressure hydrocephalus cerebrospinal fluid (CSF), co-migrating with NPFF-related synthetic peptides, were characterized by capillary HPLC coupled on-line to nanospray ion trap tandem mass spectrometry. Two peptides present in the pro-NPFF(A) precursor, NPAF (AGEGLNSQFWSLAAPQRF-NH2) and NPSF (SLAAPQRF-NH2), were identified. The monitoring of NPFF-related peptides in human CSF can be helpful to understand their roles in pain sensitivity.     

Identification of glycoproteins in human cerebrospinal fluid with a complementary proteomic approach

Biomarkers are pressingly needed to assist with the clinical diagnosis of neurodegenerative diseases and/or the monitoring of disease progression. Glycoproteins are enriched in bodily fluids such as human cerebrospinal fluid (CSF), an ideal source for discovering biomarkers due to its proximity to the central nervous system (CNS), and consequently can serve as diagnostic and/or therapeutic markers for CNS diseases. We report here an in-depth identification of glycoproteins in human CSF using a complementary proteomic approach which integrated hydrazide chemistry and lectin affinity column for glycoprotein enrichment, followed by multidimensional chromatography separation and tandem mass spectrometric analysis. Using stringent criteria, a total of 216 glycoproteins, including many low-abundance proteins, was identified with high confidence. Approximately one-third of these proteins was already known to be relevant to the CNS structurally or functionally. This investigation, for the first time, not only categorized many glycoproteins in human CSF but also expanded the existing overall CSF protein database.     

Serum procalcitonin and cerebrospinal fluid cytokines level in children with meningitis

AIMS: To determine the level of serum procalcitonin and cerebrospinal fluid cytokines in children with bacterial or viral meningitis and to document the use of these parameters in differential diagnosis. RESULTS: Before the start of antibiotic treatment, serum procalcitonin and tumor necrosis factor alpha levels were found to be higher in acute bacterial meningitis compared with viral meningitis and with the control group. Similarly, cerebrospinal fluid interleukin-6 levels were found to be significantly higher in children with acute bacterial meningitis compared with viral meningitis. However, no significant difference was determined between groups in respect to the cerebrospinal fluid interleukin-8 level. CONCLUSION: Serum procalcitonin and cerebrospinal fluid tumor necrosis factor alpha levels can be used in the early diagnosis of bacterial meningitis. Similarly, they may be useful adjuncts in differential diagnosis of bacterial and viral meningitis.     

Mechanism of prolactin release by 5-hydroxytryptophan

Male Wistar rats were intraperitoneally administered 300 mg/kg b.w. of α-methyl-p-tyrosine methyl ester(α-MT). These α-MT pretreated rats were anesthetized with urethane and then 5% glucose or dopamine (1 μg/kg b.w./min) was infused for 45 min. At 1 min before or 15 min after dopamine infusion, 10 or 50 mg/kg of 5-hydroxytryptophan (5-HTP) was injected intraperitoneally, and blood samples were taken from the jugular vein for prolactin determination. In rats treated with α-MT, the administration of 5-HTP increases the serum prolactin level in a dose-related manner. Dopamine infusion caused a marked decrease in serum prolactin level. The concomitant administration of dopamine and 5-HTP prevented the dopamine-induced decrease of serum prolactin in α-MT treated rats. These results indicate that the serotonergic stimulus enhanced prolactin release, not by inhibiting the dopaminergic activity, but by stimulating a prolactin-releasing factor or by activating other neurotransmitter systems.     

手足口病脑炎患儿脑脊液中细胞因子水平的比较

目的探讨不同病原体(CV-A6、CV-A16、EV-A71)所致手足口病脑炎患儿脑脊液中IL-6、IL-10、TNF-α、IFN-γ水平及意义。方法采用随机抽样的方法选取2018年3—5月在医院感染科收治的HFMD患儿,其中EV-A71组90例,CV-A6组77例,CV-A16组65例,选择同期高热惊厥患儿20例作为对照(高热惊厥组)。患儿入院后1～2 d行腰椎穿刺术,收集脑脊液2 mL,用流式细胞检测术分别检测细胞因子IL-6、IL-10、TNF-α、IFN-γ水平。结果 EV-A71组、CV-A6组、CV-A16组IL-6水平均明显高于高热惊厥组(t分别为6.224、7.579、6.667,P!0.05),且组间差异有统计学意义(F=18.631,P<0.05)。EV-A71组、CV-A6组、CV-A16组IL-10水平均高于高热惊厥组,差异具有统计学意义(t分别为5.387、3.227、3.084,P!0.05),且组间差异有统计学意义(F=17.480,P<0.05)。EV-A71组、CV-A6组、CV-A16组TNF-α水平与高热惊厥组差异均无统计学意义(t=-1.071、1.498、0.400,P>0.05),组间差异有统计学意义(F=6.069,P<0.05)。EV-A71组、CV-A6组、CV-A16组IFN-γ水平均高于高热惊厥组,差异具有统计学意义(t分别为4.718、7.303、8.919,P!0.05),且组间差异有统计学意义(F=16.566,P<0.05)。结论 EV-A71、CV-A6、CV-A16所致重症HFMD患儿脑脊液中IL-6、IL-10、IFN-γ均升高,表明在这3种不同病原体所致HFMD患儿脑炎中,IL-6、IL-10、IFN-γ均起到重要作用。其中,IL-6、IFN-γ明显升高,可作为疾病严重程度的评价指标。     

Quantification of Gluten Exorphin A5 in cerebrospinal fluid by liquid chromatography-mass spectrometry

In the present work, for the first time, a method for the quantification of the alimentary opioid peptide Gluten Exorphin A5 (GE-A5; Gly-Tyr-Tyr-Pro-Thr) in cerebrospinal fluid (CSF) was developed. Aliquots (5 microL) of CSF were injected into a liquid chromatography-mass spectrometry (LC-MS) instrument equipped with a reversed-phase C18 column at a flow-rate of 0.4 mL/min. The mobile phase consisted of Eluent A water with 0.6% acetic acid as an ion-pairing reagent and Eluent B acetonitrile/methanol (75:25, v/v). The LC-MS system was programmed to divert column flow to waste for 4 min after injection, after which time flow was directed into the mass spectrometer that operated in positive ion mode. No significant interfering peaks were detected at the retention times of GE-A5 in CSF blanks. The lower limit of detection and the lower limit of quantitation values for GE-A5 in CSF were established at 0.60 and 1.50 ng/mL, respectively. The intra- and inter-day precision values were <5% relative standard deviation. The intra- and inter-day accuracy were 99.6-102.8% and 100.0-101.9%, respectively. The reported assay employs extremely small volumes of CSF, thus allowing the analysis of GE-A5 from both small and large animal models.     

Intrathecal radiogold prophylaxis and the cerebrospinal fluid findings in children with acute lymphoblastic leukemia

Intrathecal radiogold application represents an alternative to the prophylaxis of meningosis in childhood acute lymphoblastic leukemia. Its compatibility is good, there are rarely any clinical side-effects. In addition to inconstant phagocytosis, changes of the protein value and its fractions could be identified in the cerebrospinal fluid. The cumulative rates of remission and survival are less marked in these groups of patients than in those who received a prophylactic skull irradiation.     

Eosinophilia in the cerebrospinal fluid of children with shunts implanted for the treatment of internal hydrocephalus

Cerebrospinal fluid (CSF) eosinophilia was observed in 26 of 404 children after the implantation of shunts for the treatment of internal hydrocephalus. High levels of 65% and 78% were recorded in two cases, which are reported in detail. In the remaining 24 cases, CSF eosinophilia ranging between 1% and 3% was found. None of the cases with CSF eosinophilia had blood eosinophilia. The cases indicate that a reaction to the material used for the shunt should be considered along with possible parasitic infestations in patients with such findings.