ABSTRACT: Data was collected on a LC-MS/MS system positive mode. Plasma and cerebrospinal fluid data from humans were acquired on the Q-Exactive with a c-18 column
Project description:Inter-individual differences in cortisol production by the hypothalamus–pituitary–adrenal (HPA) axis are thought to contribute to clinical and pathological heterogeneity of multiple sclerosis (MS). At the same time, accumulating evidence indicates that MS pathogenesis may originate in the normal-appearing white matter (NAWM). Therefore, we performed a genome-wide transcriptional analysis of post-mortem NAWM of 9 control subjects and 18 MS patients to investigate to what extent gene expression reflects disease heterogeneity and HPA-axis activity. Activity of the HPA axis was determined by cortisol levels in cerebrospinal fluid and by numbers of corticotropin-releasing neurons in the hypothalamus, while duration of MS and time to EDSS6 served as indicator of disease severity. Applying weighted gene co-expression network analysis led to the identification of a range of gene modules with highly similar co-expression patterns that strongly correlated with various indicators of HPA-axis activity and/or severity of MS. Interestingly, molecular profiles associated with relatively mild MS and high HPA-axis activity were characterized by increased expression of genes that actively regulate inflammation and by molecules involved in myelination, anti-oxidative mechanism, and neuroprotection. Additionally, group-wise comparisons of gene expression in white matter from control subjects and NAWM from (subpopulations of) MS patients uncovered disease-associated gene expression as well as strongly up- or downregulated genes in patients with relatively benign MS and/or high HPA-axis activity, with many differentially expressed genes being previously undescribed in the context of MS. Overall, the data suggest that HPA-axis activity strongly impacts on molecular mechanisms in NAWM of MS patients, but partly also independently of disease severity.
Project description:Lipids comprise 70% of the myelin sheath, and autoantibodies against lipids may contribute to the demyelination that characterizes multiple sclerosis (MS). We used lipid antigen microarrays and lipid mass spectrometry to identify bona fide lipid targets of the autoimmune response in MS brain and an animal model of MS to explore the role of the identified lipids in autoimmune demyelination. We found that autoantibodies in MS target a phosphate group in phosphatidylserine and oxidized phosphatidylcholine derivatives. Administration of these lipids ameliorated experimental autoimmune encephalomyelitis by suppressing activation and inducing apoptosis of autoreactive T cells, effects mediated by the lipids' saturated fatty-acid side chains. Thus, phospholipids represent a natural anti-inflammatory class of compounds that have potential as novel therapeutics for MS. Fig. 1A. Lipid-array profiling of IgG+IgM antibody reactivity in cerebrospinal fluid (CSF) samples from MS patients (relapsing remitting MS; secondary progressive MS; primary progressive MS), healthy controls, and other neurological disease controls. Lipid hits with the lowest FDR (q=0.048) were clustered according to their reactivity profiles. 48 different lipids were custom-spotted in duplicate using the CAMAG Automatic TLC Sampler (ATS4) robot to spray 200 nl of 10 to 100 pmol of lipids onto PVDF membranes affixed to the surface of microscope slides. The slides were probed with cerebrospinal fluid (CSF) from 59 human patient samples. 60 slides total: 18 relapsing-remitting MS, 14 secondary-progressive MS, 1 primary-progressive MS, 21 other neurological disease, 5 healthy control, 1 secondary Ab alone (not included in this submission). CSF diluted 1/10. HRP-conjugated secondary Ab (goat anti-human IgM/IgG) diluted 1/8000. ECL for 3 minutes.
Project description:FLASH-seq: plate-based full-length single-cell RNA-seq of 482 FACS index-sorted single T/B cells (CD3+/CD20+ and CD3+/CD20-) from paired cerebrospinal fluid (CSF) and PBMC of 3 MS donors (MS_13 clinically isolated syndrome, MS_14 primary progressive MS, MS_15 relapsing-remitting MS). All donors are de-identified by study code.
Project description:This SuperSeries is composed of the following subset Series: GSE37664: Human cerebrospinal fluid autoantibody lipid microarray profiling (Fig. 1A) GSE37670: Human cerebrospinal fluid autoantibody lipid microarray profiling (Fig. 2A) GSE37826: Human cerebrospinal fluid autoantibody lipid microarray profiling (Fig. 2C) Refer to individual Series
Project description:Objective: Progression in multiple sclerosis (MS) often corresponds to irreversible disability in MS patients. Cellular changes in the cerebrospinal fluid (CSF) have provided biomarkers and mechanisms in relapsing-remitting MS (RRMS) but remain understudied in primary and secondary progressive MS (summarized herein as PMS). Methods: We combined retrospective flow cytometry of CSF cells from RRMS (n = 169), PMS (n = 56), and non-inflammatory controls (n = 74) with prospective CSF single-cell transcriptomics of 35 individuals (11 controls, 12 RRMS, and 12 PMS) and with confirmatory CSF ELISA. Available CSF single cell data from age-matched and Alzheimer’s disease served as additional controls. Results: Proportions of CD14+ monocytes in CSF are increased in PMS and correlated with clinical surrogate markers of progression. Transcriptionally, these monocytes resembled border-associated macrophages (BAM)-like cells with a chronically activated antigen-presenting phenotype. Additionally, these monocytes shared some features with disease-associated microglia/macrophages (DAM), previously identified in neurodegeneration. Induction of DAM-associated molecules, including transcribed and soluble TREM2, was unique to SPMS and supported its differential diagnosis. Interpretation: We thus identified MS stage-specific CSF signatures and shared cellular features of degeneration detectable in CSF of PMS patients.
Project description:Objective: Progression in multiple sclerosis (MS) often corresponds to irreversible disability in MS patients. Cellular changes in the cerebrospinal fluid (CSF) have provided biomarkers and mechanisms in relapsing-remitting MS (RRMS) but remain understudied in primary and secondary progressive MS (summarized herein as PMS). Methods: We combined retrospective flow cytometry of CSF cells from RRMS (n = 169), PMS (n = 56), and non-inflammatory controls (n = 74) with prospective CSF single-cell transcriptomics of 35 individuals (11 controls, 12 RRMS, and 12 PMS) and with confirmatory CSF ELISA. Available CSF single cell data from age-matched and Alzheimer’s disease served as additional controls. Results: Proportions of CD14+ monocytes in CSF are increased in PMS and correlated with clinical surrogate markers of progression. Transcriptionally, these monocytes resembled border-associated macrophages (BAM)-like cells with a chronically activated antigen-presenting phenotype. Additionally, these monocytes shared some features with disease-associated microglia/macrophages (DAM), previously identified in neurodegeneration. Induction of DAM-associated molecules, including transcribed and soluble TREM2, was unique to SPMS and supported its differential diagnosis. Interpretation: We thus identified MS stage-specific CSF signatures and shared cellular features of degeneration detectable in CSF of PMS patients.
Project description:B cell-depleting therapies are effective in multiple sclerosis (MS), yet many patients progress toward disability, highlighting the need for improved interventions. To identify new therapeutic targets, we generated a single-cell RNA-seq atlas of cerebrospinal fluid (CSF), brain, and blood from non-inflammatory controls and patients with MS or other neuroinflammatory diseases. We found disease-associated enrichment of class-switched IgG⁺ B cells and plasma cells in the CSF of patients with MS. Unbiased analysis revealed a rare subset of activated PD-1⁺, TCR-restricted, B-cell recruiting T follicular helper-like cells enriched in MS CSF. To therapeutically target this population, we developed PD-1-directed CAR T cells that selectively depleted pathogenic PD-1⁺ CD4 T cells. In a murine model of neuroinflammation, this approach attenuated CNS inflammation and improved clinical outcomes, particularly when using an IL-10-armored CAR construct. These findings establish a cellular framework for CNS-localized adaptive immunity in MS and outline a strategy for its selective disruption.
Project description:We performed shallow whole genome sequencing (WGS) on circulating free (cf)DNA extracted from plasma or cerebrospinal fluid (CSF), and shallow WGS on the tissue DNA extracted from the biopsy in order to evaluate the correlation between the two biomaterials. After library construction and sequencing (Hiseq3000 or Ion Proton), copy number variations were called with WisecondorX.
Project description:Proteome analysis of cerebrospinal fluid is associated with many of the same challenges that has to be faced when analyzing other biofluids like plasma or urine. Most importantly is the high dynamic range of protein concentrations, which makes it difficult to quantify more than a thousand proteins in a singleshot analysis of a tryptic digest of a raw sample. In addition, cerebrospinal fluid—especially when collected from children—is a much more scarce and precious sample as compare to urine and plasma. The aim of this work therefore was to find the optimal way to process and analyze cerebrospinal fluid from a minimal amount of sample and still obtain the deepest possible cerebrospinal fluid proteome. The work shows that enrichment of extracellular vesicles by ultracentrifugation increases the complexity of the cerebrospinal fluid proteome by two-fold or more. Thus, by following the enrichment protocol, more than two thousand proteins can routinely be quantified reproducibly by label-free quantification and data independent acquisition (DIA) in single-shot LC-MSMS runs of less than one hour. The project also present an optimized workflow for cerebrospinal fluid proteomics that enables a high-throughput method with a short chromatographic gradient (12 min) allowing analysis of hundred samples per day and still obtaining deep proteome coverage, especially when including a study-specific spectral library generated by repeated injection and gas-phase fractionation of pooled samples.