Liquid biopsies in epilepsy: biomarkers for etiology, diagnosis, prognosis, and therapeutics.

Epilepsy is one of the most common diseases of the central nervous system, impacting nearly 50 million people around the world. Heterogeneous in nature, epilepsy presents in children and adults alike. Currently, surgery is one treatment approach that can completely cure epilepsy. However, not all individuals are eligible for surgical procedures or have successful outcomes. In addition to surgical approaches, antiepileptic drugs (AEDs) have also allowed individuals with epilepsy to achieve freedom from seizures. Others have found treatment through nonpharmacologic approaches such as vagus nerve stimulation, or responsive neurostimulation. Difficulty in accessing samples of human brain tissue along with advances in sequencing technology have driven researchers to investigate sampling liquid biopsies in blood, serum, plasma, and cerebrospinal fluid within the context of epilepsy. Liquid biopsies provide minimal or non-invasive sample collection approaches and can be assayed relatively easily across multiple time points, unlike tissue-based sampling. Various efforts have investigated circulating nucleic acids from these samples including microRNAs, cell-free DNA, transfer RNAs, and long non-coding RNAs. Here, we review nucleic acid-based liquid biopsies in epilepsy to improve understanding of etiology, diagnosis, prediction, and therapeutic monitoring.

DNA versus RNA oxidation in Parkinson's disease: Which is more important?

BACKROUND: 8-hydroxy-2 deoxyguanosine (8-OHdG) and the 8-hydroxyguanosine (8-OHG) are the most widely used biomarkers of nucleoside oxidation affecting DNA and RNA and are considered reliable markers of oxidative stress. Increased levels of these markers are found in the various biological fluids of patients with neurodegenerative disorders. OBJECTIVE: The primary aim of our study was to assess the differences of investigated markers between patient groups and subsequently study the influence of clinical factors that might modify the levels of investigated markers during the disease progression. METHODS: In this study, we analysed the 8-OHdG and 8-OHG levels in the cerebrospinal fluid (CSF) and serum from 44 patients with Parkinson's disease (PD) and 32 controls using an ELISA. RESULTS: There were significantly higher CSF levels of both investigated markers in Parkinson's disease patients as compared to controls (p=0.02 and p=0.04). Significantly higher CSF values of 8-OHdG were found in PD patients without dementia (p=0.05), whereas patients with dementia recorded lower 8-OHG CSF levels compared to controls (p=0.04). The disease duration and age influenced the levels of both markers within investigated groups. CONCLUSION: Oxidative DNA damage plays an important role in the early stages of PD, whereas during the progression of the disease the process is more complex, and other mechanisms are in the foreground. The measurement of 8-OHdG might be used as an "early-stage marker", whereas the decrease of 8-OHG in CSF might reflect the degree of neurodegeneration during the disease progression, suggesting its utility as a prognostic marker of advanced PD stages.

Soluble membrane attack complex in the blood and cerebrospinal fluid of HIV-infected individuals, relationship to HIV RNA, and comparison with HIV negatives.

The soluble membrane attack complex (sMAC) represents the terminal product of the complement cascade. We enrolled 47 HIV+ adults (12 of whom underwent a second visit at least 24weeks after starting therapy) as well as 11 HIV negative controls. At baseline, cerebrospinal fluid (CSF) sMAC was detectable in 27.7% of HIV+ individuals. CSF sMAC correlated with CSF HIV RNA levels and was more likely to be detectable in HIV+ individuals on cART compared to HIV negative controls. In HIV+ participants, there were negative association trends between sMAC and neurocognitive performance but these did not reach statistical significance.

Blood-CSF barrier and compartmentalization of CNS cellular immune response in HIV infection.

HIV infection is persistent in the CNS, to evaluate the compartmentalization of the CNS immune response to HIV, we compared soluble markers of cellular immunity in the blood and CSF among HIV- (n=19) and HIV+ (n=68), as well as among HIV participants with or without CSF pleocytosis. Dysfunction of the blood cerebrospinal fluid barrier (BCSFB) was common in HIV participants. CSF levels of TNFα, IFNγ, IL-2, IL-6, IL-7, IL-10, IP-10, MIP-1α, MIP-1ß, and RANTES were significantly higher in participants with CSF pleocytosis (P<0.05); serum levels of these biomarkers were comparable. The CNS immune response is compartmentalized, and remains so despite the BCSFB dysfunction during HIV infection; it is markedly reduced by virology suppression, although BCSFB dysfunction persists on this subgroup.

Transcriptomic Profiling of Extracellular RNAs Present in Cerebrospinal Fluid Identifies Differentially Expressed Transcripts in Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is a debilitating neurological disorder for which prognostic and diagnostic biomarkers are lacking. Cerebrospinal fluid (CSF) is an accessible body fluid that comes into direct contact with the central nervous system (CNS) and acts as a nuclease-free repository where RNA transcripts shed by brain tissues can reside for extended periods of time. OBJECTIVE: We studied the RNA species present in the CSF of PD patients to identify novel diagnostic biomarkers. METHODS: Small volumes of CSF from 27 PD patients and 30 healthy age- and sex-matched controls were used for RNA extraction followed by next-generation sequencing (RNA-seq) using the Illumina platform. CSF contains a number of fragmented RNA species that were individually sequenced and analyzed. Comparing PD to control subjects, we observed a pool of dysregulated sequencing tags that were further analyzed and validated by quantitative real-time PCR (qRT-PCR). RESULTS: A total of 201 differentially expressed sequencing tags (DETs), including 92 up-regulated and 109 down-regulated DETs were identified. We validated the following DETs by real time PCR in the patient samples: Dnmt1, Ezh2, CCR3, SSTR5,PTPRC, UBC, NDUFV2, BMP7, SCN9, SCN9 antisense (AC010127.3), and long noncoding RNAs AC079630 and UC001lva.4 (close to the LRRK2 gene locus), as potential PD biomarkers. CONCLUSIONS: The CSF is a unique environment that contains many species of RNA. Our work demonstrates that CSF can potentially be used to identify biomarkers for the detection and tracking of disease progression and evaluation of therapeutic outcomes.

The landscape of microRNA, Piwi-interacting RNA, and circular RNA in human saliva.

BACKGROUND: Extracellular RNAs (exRNAs) in human body fluids are emerging as effective biomarkers for detection of diseases. Saliva, as the most accessible and noninvasive body fluid, has been shown to harbor exRNA biomarkers for several human diseases. However, the entire spectrum of exRNA from saliva has not been fully characterized. METHODS: Using high-throughput RNA sequencing (RNA-Seq), we conducted an in-depth bioinformatic analysis of noncoding RNAs (ncRNAs) in human cell-free saliva (CFS) from healthy individuals, with a focus on microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), and circular RNAs (circRNAs). RESULTS: Our data demonstrated robust reproducibility of miRNA and piRNA profiles across individuals. Furthermore, individual variability of these salivary RNA species was highly similar to those in other body fluids or cellular samples, despite the direct exposure of saliva to environmental impacts. By comparative analysis of >90 RNA-Seq data sets of different origins, we observed that piRNAs were surprisingly abundant in CFS compared with other body fluid or intracellular samples, with expression levels in CFS comparable to those found in embryonic stem cells and skin cells. Conversely, miRNA expression profiles in CFS were highly similar to those in serum and cerebrospinal fluid. Using a customized bioinformatics method, we identified >400 circRNAs in CFS. These data represent the first global characterization and experimental validation of circRNAs in any type of extracellular body fluid. CONCLUSIONS: Our study provides a comprehensive landscape of ncRNA species in human saliva that will facilitate further biomarker discoveries and lay a foundation for future studies related to ncRNAs in human saliva.

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.

Cerebrospinal fluid markers that predict SIV CNS disease.

Predictive cerebrospinal fluid markers would provide valuable tools for tracking the development and progression of HIV CNS disease. In this study, expression of IL-6, MCP-1, and viral RNA in cerebrospinal fluid collected from SIV-inoculated macaques during acute, asymptomatic, and terminal stages of infection was quantitated to determine whether one or several of these parameters paralleled the severity of SIV encephalitis. Animals that developed moderate to severe SIV encephalitis had significantly elevated levels of CSF IL-6, MCP-1, and SIV RNA during asymptomatic infection and persisting through terminal disease as compared to animals developing mild or no CNS disease.

Rotavirus encephalopathy: pathogenesis reviewed.

Two cases of rotavirus gastroenteritis associated with neurological involvement, one with encephalitis (defined by abnormal neurological signs, cerebrospinal fluid (CSF) pleocytosis and detection of rotavirus genomic nucleic acid in the CSF) and one with a non-inflammatory encephalopathy (defined by abnormal neurological signs, an entirely normal CSF and detection of rotavirus genomic nucleic acid in the CSF), are presented and used as a basis to review and explore potential pathogenetic mechanisms, including direct viral replication within neurons and indirect effects of the newly described rotavirus 'enterotoxin'.

Detection of in vivo stimulated cerebrospinal-fluid lymphocytes by flow cytometry in patients with multiple sclerosis.

To examine the immune response in the central nervous system in patients with multiple sclerosis (MS), we characterized the cell-cycle phase of lymphocytes from cerebrospinal fluid. Cells were stained with acridine orange, and both RNA and DNA content were determined by flow cytometry. Although most cells were in the quiescent phase of the cycle, a significant increase was seen in the percentage of cells in the first stage of the proliferative cycle (G1 phase) when 21 samples of cerebrospinal fluid from 17 patients with MS were compared with samples from 21 controls (P < 0.001). Stimulated cells in the first stage of the cycle were seen in all categories of MS: active, progressive, or stable. In addition, increased numbers of cells in the second stage of the cycle (S phase) were seen in six specimens from patients with MS, five of whom had actie disease, but no increases were seen in controls (P < 0.02). These data indicate that stimulated lymphocytes were present in the cerebrospinal fluid durng all phases of MS and that stimulation becomes more intense during an acute exacerbation.