Differential gene expression in B cells and T helper cells following high-dose glucocorticoid therapy for multiple sclerosis relapse.

BACKGROUND: Despite remarkable advances in the therapy of multiple sclerosis (MS), patients with MS may still experience relapses. High-dose short-term methylprednisolone (MP) remains the standard treatment in the acute management of MS relapses due to its potent anti-inflammatory and immunosuppressive properties. However, there is a lack of studies on the cell type-specific transcriptome changes that are induced by this synthetic glucocorticoid (GC). Moreover, it is not well understood why some patients do not benefit adequately from MP therapy. METHODS: We collected peripheral blood from MS patients in relapse immediately before and after ∼3-5 days of therapy with MP at 4 study centers. CD19+ B cells and CD4+ T cells were then isolated for profiling the transcriptome with high-density arrays. The patients' improvement of neurological symptoms was evaluated after ∼2 weeks by the treating physicians. We finally analyzed the data to identify genes that were differentially expressed in response to the therapy and whose expression differed between clinical responders and non-responders. RESULTS: After MP treatment, a total of 33 genes in B cells and 55 genes in T helper cells were significantly up- or downregulated. The gene lists overlap in 10 genes and contain genes that have already been described as GC-responsive genes in the literature on other cell types and diseases. Their differential expression points to a rapid and coordinated modulation of multiple signaling pathways that influence transcription. Genes that were previously suggested as potential prognostic biomarkers of the clinical response to MP therapy could not be confirmed in our data. However, a greater increase in the expression of genes encoding proteins with antimicrobial activity was detected in CD4+ T cells from non-responders compared to responders. CONCLUSION: Our study delved into the cell type-specific effects of MP at the transcriptional level. The data suggest a therapy-induced ectopic expression of some genes (e.g., AZU1, ELANE and MPO), especially in non-responders. The biological consequences of this remain to be explored in greater depth. A better understanding of the molecular mechanisms underlying clinical recovery from relapses in patients with MS will help to optimize future treatment decisions.

Transcriptome alterations in peripheral blood B cells of patients with multiple sclerosis receiving immune reconstitution therapy.

BACKGROUND: Multiple sclerosis (MS) is a chronic, inflammatory and neurodegenerative disease that leads to irreversible damage to the brain and spinal cord. The goal of so-called "immune reconstitution therapies" (IRTs) is to achieve long-term disease remission by eliminating a pathogenic immune repertoire through intense short-term immune cell depletion. B cells are major targets for effective immunotherapy in MS. OBJECTIVES: The aim of this study was to analyze the gene expression pattern of B cells before and during IRT (i.e., before B-cell depletion and after B-cell repopulation) to better understand the therapeutic effects and to identify biomarker candidates of the clinical response to therapy. METHODS: B cells were obtained from blood samples of patients with relapsing-remitting MS (n = 50), patients with primary progressive MS (n = 13) as well as healthy controls (n = 28). The patients with relapsing MS received either monthly infusions of natalizumab (n = 29) or a pulsed IRT with alemtuzumab (n = 15) or cladribine (n = 6). B-cell subpopulation frequencies were determined by flow cytometry, and transcriptome profiling was performed using Clariom D arrays. Differentially expressed genes (DEGs) between the patient groups and controls were examined with regard to their functions and interactions. We also tested for differences in gene expression between patients with and without relapse following alemtuzumab administration. RESULTS: Patients treated with alemtuzumab or cladribine showed on average a > 20% lower proportion of memory B cells as compared to before IRT. This was paralleled by profound transcriptome shifts, with > 6000 significant DEGs after adjustment for multiple comparisons. The top DEGs were found to regulate apoptosis, cell adhesion and RNA processing, and the most highly connected nodes in the network of encoded proteins were ESR2, PHB and RC3H1. Higher mRNA levels of BCL2, IL13RA1 and SLC38A11 were seen in patients with relapse despite IRT, though these differences did not pass the false discovery rate correction. CONCLUSIONS: We show that B cells circulating in the blood of patients with MS undergoing IRT present a distinct gene expression signature, and we delineated the associated biological processes and gene interactions. Moreover, we identified genes whose expression may be an indicator of relapse risk, but further studies are needed to verify their potential value as biomarkers.

Correlation of Nabiximols Dose to Steady-State Concentrations of Cannabinoids in Urine Samples from Patients with Multiple Sclerosis.

Therapeutic drug monitoring of Δ9-tetrahydrocannabinol (THC) and cannabidiol (CBD) is based on a complex procedure and is therefore not possible in most laboratories, especially in emergency cases. This work addresses the question of whether therapeutic drug monitoring of nabiximols can be performed using an immunological urine-based test system for cannabinoid abuse. Seventeen patients with multiple sclerosis were included in this study. Administered doses of nabiximols were correlated with immunologically determined urine concentrations of cannabinoids using the DRITM Cannabinoid (THC) Assay. Significant correlations with the administered nabiximols doses were found for creatinine-normalized urine concentrations of cannabinoids without (r = 0.675; p = 0.0015) and after (r = 0.650; p = 0.0044) hydrolysis, as well as for gas-chromatography-coupled mass spectrometry (GC/MS)-measured concentrations of the THC metabolite 11-nor-9-carboxy-Δ9-THC (THC-COOH) in urine samples (r = 0.571; p = 0.0084) by Pearson's correlation. In addition, doses were significantly correlated with plasma THC-COOH concentrations (r = 0.667; p = 0.0017) measured by GC/MS. Simple immunological cannabinoid measurements in urine samples could provide an estimate of nabiximols dosage, although the correlations obtained here were weak because of the small number of patients observed. Longitudinal monitoring of individual patients is expected to exhibit good results of therapeutic drug monitoring of nabiximols.

Implication of genetic variants in primary microRNA processing sites in the risk of multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system with a well-established genetic contribution to susceptibility. Over 200 genetic regions have been linked to the inherited risk of developing MS, but the disease-causing variants and their functional effects at the molecular level are still largely unresolved. We hypothesised that MS-associated single-nucleotide polymorphisms (SNPs) affect the recognition and enzymatic cleavage of primary microRNAs (pri-miRNAs). METHODS: Our study focused on 11 pri-miRNAs (9 primate-specific) that are encoded in genetic risk loci for MS. The levels of mature miRNAs and potential isoforms (isomiRs) produced from those pri-miRNAs were measured in B cells obtained from the peripheral blood of 63 MS patients and 28 healthy controls. We tested for associations between SNP genotypes and miRNA expression in cis using quantitative trait locus (cis-miR-eQTL) analyses. Genetic effects on miRNA stem-loop processing efficiency were verified using luciferase reporter assays. Potential direct miRNA target genes were identified by transcriptome profiling and computational binding site assessment. FINDINGS: Mature miRNAs and isomiRs from hsa-mir-26a-2, hsa-mir-199a-1, hsa-mir-4304, hsa-mir-4423, hsa-mir-4464 and hsa-mir-4492 could be detected in all B-cell samples. When MS patient subgroups were compared with healthy controls, a significant differential expression was observed for miRNAs from the 5' and 3' strands of hsa-mir-26a-2 and hsa-mir-199a-1. The cis-miR-eQTL analyses and reporter assays pointed to a slightly more efficient Drosha-mediated processing of hsa-mir-199a-1 when the MS risk allele T of SNP rs1005039 is present. On the other hand, the MS risk allele A of SNP rs817478, which substitutes the first C in a CNNC sequence motif, was found to cause a markedly lower efficiency in the processing of hsa-mir-4423. Overexpression of hsa-mir-199a-1 inhibited the expression of 60 protein-coding genes, including IRAK2, MIF, TNFRSF12A and TRAF1. The only target gene identified for hsa-mir-4423 was TMEM47. INTERPRETATION: We found that MS-associated SNPs in sequence determinants of pri-miRNA processing can affect the expression of mature miRNAs. Our findings complement the existing literature on the dysregulation of miRNAs in MS. Further studies on the maturation and function of miRNAs in different cell types and tissues may help to gain a more detailed functional understanding of the genetic basis of MS. FUNDING: This study was funded by the Rostock University Medical Center (FORUN program, grant: 889002), Sanofi Genzyme (grant: GZ-2016-11560) and Merck Serono GmbH (Darmstadt, Germany, an affiliate of Merck KGaA, CrossRef Funder ID: 10.13039/100009945, grant: 4501860307). NB was supported by the Stiftung der Deutschen Wirtschaft (sdw) and the FAZIT foundation. EP was supported by the Landesgraduiertenförderung Mecklenburg-Vorpommern.

Rituximab versus mitoxantrone: comparing effectiveness and safety in advanced relapsing multiple sclerosis.

BACKGROUND: Rituximab (RTX), a CD20 depleting agent, is a frequently used off-label treatment for multiple sclerosis (MS), while mitoxantrone (MTX) is approved, albeit rarely used for active relapsing MS (RMS). However, observational data comparing RTX and MTX effectiveness and safety are scarce. OBJECTIVE: We aimed to compare effectiveness and safety of MTX and RTX in patients with active RMS. METHODS: From combined retrospective clinical data of three MS centers, we selected patients who had received at least one infusion of RTX or MTX and had at least a 6-month clinical follow-up available. Treatment groups were compared by propensity score (PS)-adjusted regression and inverse PS-weighted generalized estimated equation models regarding disability progression, relapse activity, and adverse events (AEs). RESULTS: We included 292 RMS patients (mean age 41.8 years, 71.6% female) who received RTX (119 patients, mean age 36.8 years, 74.8% female) or MTX (173 patients mean age 45.3 years, 69.4% female). Using both PS methods, we did not find a significant effect favoring RTX or MTX treatment regarding the probability of disability worsening or relapse occurrence. However, RTX treatment was associated with a significantly lower probability of severe AEs and AEs. CONCLUSIONS: RTX shows comparable effectiveness but a favorable safety profile compared with MTX in active RMS.

Therapeutic plasma exchange in steroid-refractory multiple sclerosis relapses. A retrospective two-center study.

BACKGROUND: Therapeutic plasma exchange (TPE) is frequently used in glucocorticosteroid (GCS)-refractory multiple sclerosis (MS) relapses. Data regarding predictors of treatment response are scarce. The objective of this study was to analyze predictive factors for response to TPE in GCS-refractory MS patients. METHODS: A total of 118 MS patients in two tertiary MS centers were analyzed. Primary outcome was TPE response defined as marked, mild, or no improvement. Secondary outcome was change in expanded disability status scale (ΔEDSS). ΔEDSS and relapse activity within 6 months after TPE were studied. RESULTS: Marked or mild improvement was observed in 78.8% of patients. ΔEDSS correlated significantly inversely with time from relapse to start of TPE (τ = -0.239, p = 0.001), age (τ = 0.182, p = 0.009) and disease duration (τ = -0.167, p = 0.017). In multivariate analysis, TPE response was predicted by diagnosis of relapsing MS [odds ratio (OR): 3.1], gadolinum-enhancement on magnetic resonance imaging (OR 3.2), age (OR 0.5 per 5 years older) and time from relapse onset to TPE (OR 0.7 per 7 days longer). CONCLUSION: Patients with longer disease duration and higher EDSS pre and post-TPE were more likely to show further disability progression or relapses within 6 months after TPE. No sustained effects were observed during the follow-up period.

Molecularization of Bitter Off-Taste Compounds in Pea-Protein Isolates (Pisum sativum L.).

Activity-guided fractionations, combined with taste dilution analyses (TDA), were performed to locate the key compounds contributing to the bitter off-taste of pea-protein isolates (Pisum sativum L.). Purification of the compounds perceived with the highest sensory impact, followed by 1D/2D-NMR, (LC-)MS/MS, LC-TOF-MS, and MSE experiments, led to the identification of 14 lipids and lipid oxidation products, namely, 9,10,13-trihydroxyoctadec-12-enoic acid, 9,12,13-trihydroxyoctadec-10-enoic acid, 9,10,11-trihydroxyoctadec-12-enoic, 11,12,13-trihydroxyoctadec-9-enoic acid, (10E,12E)-9-hydroxyoctadeca-10,12-dienoic acid, (9Z,11E)-13-hydroxyoctadeca-9,11-dienoic acid, (9E,11E)-13-hydroxyoctadeca-9,11-dienoic acid, 1-linoleoyl glycerol, α-linolenic acid, 2-hydroxypalmitic acid, 2-hydroxyoleic acid, linoleic acid, (9Z,11E)-13-oxooctadeca-9,11-dienoic acid, and octacosa-6,9,19,22-tetraen. Herein, we present the isolation, structure determination, and sensory activity of these molecules. Depending on their structure, the isolated compounds showed human bitter recognition thresholds between 0.06 and 0.99 mmol/L in water.

A genetic variant associated with multiple sclerosis inversely affects the expression of CD58 and microRNA-548ac from the same gene.

Genome-wide association studies have identified more than 200 genetic variants to be associated with an increased risk of developing multiple sclerosis (MS). Still, little is known about the causal molecular mechanisms that underlie the genetic contribution to disease susceptibility. In this study, we investigated the role of the single-nucleotide polymorphism (SNP) rs1414273, which is located within the microRNA-548ac stem-loop sequence in the first intron of the CD58 gene. We conducted an expression quantitative trait locus (eQTL) analysis based on public RNA-sequencing and microarray data of blood-derived cells of more than 1000 subjects. Additionally, CD58 transcripts and mature hsa-miR-548ac molecules were measured using real-time PCR in peripheral blood samples of 32 MS patients. Cell culture experiments were performed to evaluate the efficiency of Drosha-mediated stem-loop processing dependent on genotype and to determine the target genes of this underexplored microRNA. Across different global populations and data sets, carriers of the MS risk allele showed reduced CD58 mRNA levels but increased hsa-miR-548ac levels. We provide evidence that the SNP rs1414273 might alter Drosha cleavage activity, thereby provoking partial uncoupling of CD58 gene expression and microRNA-548ac production from the shared primary transcript in immune cells. Moreover, the microRNA was found to regulate genes, which participate in inflammatory processes and in controlling the balance of protein folding and degradation. We thus uncovered new regulatory implications of the MS-associated haplotype of the CD58 gene locus, and we remind that paradoxical findings can be encountered in the analysis of eQTLs upon data aggregation. Our study illustrates that a better understanding of RNA processing events might help to establish the functional nature of genetic variants, which predispose to inflammatory and neurological diseases.

Transcriptome profiling of peripheral blood immune cell populations in multiple sclerosis patients before and during treatment with a sphingosine-1-phosphate receptor modulator.

AIMS: Fingolimod is a sphingosine-1-phosphate (S1P) receptor modulator approved for the treatment of the relapsing form of multiple sclerosis (MS). It prevents the egress of lymphocyte subpopulations from lymphoid tissues into the circulation. Here, we explored the broad effects of fingolimod on gene expression in different immune cell subsets. METHODS: Utilizing 150 high-resolution microarrays from Affymetrix, we obtained the transcriptome profiles of 5 cell populations, which were separated from the peripheral blood of MS patients prior to and following oral administration of fingolimod. RESULTS: After 3 months of treatment, significant transcriptome shifts were seen in CD4+ and CD8+ cells, which is mainly attributable to the selective homing of naive T cells and central memory T cells. Although the number of B cells was greatly reduced in the blood of fingolimod-treated MS patients, the analysis of differential expression in CD19+ cells identified only a small set of 42 genes, which indicated a slightly higher frequency of transitional B cells. The transcriptome signatures of CD14+ monocytes and CD56+ natural killer cells were not affected. CONCLUSION: Our study corroborates changes in the composition of circulating immune cells in response to fingolimod and delineates the respective implications at the RNA level. Our data may be valuable for comparing the effects of novel S1P receptor modulating agents, which may be a therapeutic option for patients with secondary progressive MS as well.

High-Resolution Expression Profiling of Peripheral Blood CD8+ Cells in Patients with Multiple Sclerosis Displays Fingolimod-Induced Immune Cell Redistribution.

Fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, is an oral drug approved for the treatment of active relapsing-remitting multiple sclerosis (RRMS). It selectively inhibits the egress of lymphocytes from lymph nodes. We studied the changes in the transcriptome of peripheral blood CD8+ cells to unravel the effects at the molecular level during fingolimod therapy. We separated CD8+ cells from the blood of RRMS patients before the first dose of fingolimod as well as 24 h and 3 months after the start of therapy. Changes in the expression of coding and non-coding genes were measured with high-density Affymetrix Human Transcriptome Array (HTA) 2.0 microarrays. Differentially expressed genes in response to therapy were identified by t test and fold change and analyzed for their functions and molecular interactions. No gene was expressed at significantly higher or lower levels 24 h after the first administration of fingolimod compared to baseline. However, after 3 months of therapy, 861 transcripts were found to be differentially expressed, including interleukin and chemokine receptors. Some of the genes are associated to the S1P pathway, such as the receptor S1P5 and the kinase MAPK1, which were significantly increased in expression. The fingolimod-induced transcriptome changes reflect a shift in the proportions of CD8+ T cell subsets, with CCR7- effector memory T cells being relatively increased in frequency in the blood of fingolimod-treated patients. In consequence, CCR7 mRNA levels were reduced by >80 % and genes involved in T cell activation and lymphocyte cytotoxicity were increased in expression. Gene regulatory programs caused by downstream S1P signaling had only minor effects.

The presence of FCGR2B promoter or transmembrane region variant alleles leads to reduced serum IL-6 levels in rheumatoid arthritis.

The inhibitory FcγRIIB plays an important role for the peripheral B cell tolerance and plasma cell homeostasis, and any malfunctioning is predicted to result in humoral autoimmunity. An association between rheumatoid arthritis (RA) and FCGR2B promoter and TM region variant alleles, both of which result in a reduced functionality, is only insufficiently elucidated. We here set out to investigate the impact of these variants on disease progression in European RA patients. One hundred and five ACPA-positive RA patients were genotyped for the FCGR2B -386G>C promoter and the 695T>C transmembrane region variants. Moreover, serum titers for IL-6, TNFα, CTX-1 and ACPAs were measured and peripheral blood T cell and B cell populations analyzed for expression of the activation markers CTLA-4 and CD86. The presence of an FCGR2B variant allele results in reduced serum IL-6, a trend toward later disease onset and reduced requirement for biological treatment, but does not seem to aggravate RA. Likewise, the presence of the TM region variant allele is associated with a lower activation state of the Tregs and of naïve and memory B cells. The observation of a malfunctioning FcγRIIb not aggravating RA is counterintuitive at first. However, the etiology of RA is linked to inflammatory episodes, and the lack of B cell inhibition may support an accelerated antibody-mediated clearance of the disease initiating and perpetuating agents. It would thereby shorten inflammatory episodes, postpone the onset of disease and result in a less severe course of RA in carriers of FCGR2B variant alleles.