The Utility of Miniaturized Adsorbers in Exploring the Cellular and Molecular Effects of Blood Purification: A Pilot Study with a Focus on Immunoadsorption in Multiple Sclerosis.

Immunoadsorption (IA) has proven to be clinically effective in the treatment of steroid-refractory multiple sclerosis (MS) relapses, but its mechanism of action remains unclear. We used miniaturized adsorber devices with a tryptophan-immobilized polyvinyl alcohol (PVA) gel sorbent to mimic the IA treatment of patients with MS in vitro. The plasma was screened before and after adsorption with regard to disease-specific mediators, and the effect of the IA treatment on the migration of neutrophils and the integrity of the endothelial cell barrier was tested in cell-based models. The in vitro IA treatment with miniaturized adsorbers resulted in reduced plasma levels of cytokines and chemokines. We also found a reduced migration of neutrophils towards patient plasma treated with the adsorbers. Furthermore, the IA-treated plasma had a positive effect on the endothelial cell barrier's integrity in the cell culture model. Our findings suggest that IA results in a reduced infiltration of cells into the central nervous system by reducing leukocyte transmigration and preventing blood-brain barrier breakdown. This novel approach of performing in vitro blood purification therapies on actual patient samples with miniaturized adsorbers and testing their effects in cell-based assays that investigate specific hypotheses of the pathophysiology provides a promising platform for elucidating the mechanisms of action of those therapies in various diseases.

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.

Genetic risk variants for multiple sclerosis are linked to differences in alternative pre-mRNA splicing.

Background: Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system to which a genetic predisposition contributes. Over 200 genetic regions have been associated with increased disease risk, but the disease-causing variants and their functional impact at the molecular level are mostly poorly defined. We hypothesized that single-nucleotide polymorphisms (SNPs) have an impact on pre-mRNA splicing in MS. Methods: Our study focused on 10 bioinformatically prioritized SNP-gene pairs, in which the SNP has a high potential to alter alternative splicing events (ASEs). We tested for differential gene expression and differential alternative splicing in B cells from MS patients and healthy controls. We further examined the impact of the SNP genotypes on ASEs and on splice isoform expression levels. Novel genotype-dependent effects on splicing were verified with splicing reporter minigene assays. Results: We were able to confirm previously described findings regarding the relation of MS-associated SNPs with the ASEs of the pre-mRNAs from GSDMB and SP140. We also observed an increased IL7R exon 6 skipping when comparing relapsing and progressive MS patients to healthy subjects. Moreover, we found evidence that the MS risk alleles of the SNPs rs3851808 (EFCAB13), rs1131123 (HLA-C), rs10783847 (TSFM), and rs2014886 (TSFM) may contribute to a differential splicing pattern. Of particular interest is the genotype-dependent exon skipping of TSFM due to the SNP rs2014886. The minor allele T creates a donor splice site, resulting in the expression of the exon 3 and 4 of a short TSFM transcript isoform, whereas in the presence of the MS risk allele C, this donor site is absent, and thus the short transcript isoform is not expressed. Conclusion: In summary, we found that genetic variants from MS risk loci affect pre-mRNA splicing. Our findings substantiate the role of ASEs with respect to the genetics of MS. Further studies on how disease-causing genetic variants may modify the interactions between splicing regulatory sequence elements and RNA-binding proteins can help to deepen our understanding of the genetic susceptibility to MS.

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.

Genetic, Environmental and Lifestyle Determinants of Accelerated Telomere Attrition as Contributors to Risk and Severity of Multiple Sclerosis.

Telomeres are protective structures at the ends of linear chromosomes. Shortened telomere lengths (TL) are an indicator of premature biological aging and have been associated with a wide spectrum of disorders, including multiple sclerosis (MS). MS is a chronic inflammatory, demyelinating and neurodegenerative disease of the central nervous system. The exact cause of MS is still unclear. Here, we provide an overview of genetic, environmental and lifestyle factors that have been described to influence TL and to contribute to susceptibility to MS and possibly disease severity. We show that several early-life factors are linked to both reduced TL and higher risk of MS, e.g., adolescent obesity, lack of physical activity, smoking and vitamin D deficiency. This suggests that the mechanisms underlying the disease are connected to cellular aging and senescence promoted by increased inflammation and oxidative stress. Additional prospective research is needed to clearly define the extent to which lifestyle changes can slow down disease progression and prevent accelerated telomere loss in individual patients. It is also important to further elucidate the interactions between shared determinants of TL and MS. In future, cell type-specific studies and advanced TL measurement methods could help to better understand how telomeres may be causally involved in disease processes and to uncover novel opportunities for improved biomarkers and therapeutic interventions in MS.

Systematic Review of Studies on Telomere Length in Patients with Multiple Sclerosis.

Telomeres are protective cap structures at the end of chromosomes that are essential for maintaining genomic stability. Accelerated telomere shortening is related to premature cellular senescence. Shortened telomere lengths (TL) have been implicated in the pathogenesis of various chronic immune-mediated and neurological diseases. We aimed to systematically review the current literature on the association of TL as a measure of biological age and multiple sclerosis (MS). A comprehensive literature search was conducted to identify original studies that presented data on TL in samples from persons with MS. Quantitative and qualitative information was extracted from the articles to summarize and compare the studies. A total of 51 articles were screened, and 7 of them were included in this review. In 6 studies, average TL were analyzed in peripheral blood cells, whereas in one study, bone marrow-derived cells were used. Four of the studies reported significantly shorter leukocyte TL in at least one MS subtype in comparison to healthy controls (p=0.003 in meta-analysis). Shorter telomeres in patients with MS were found to be associated, independently of age, with greater disability, lower brain volume, increased relapse rate and more rapid conversion from relapsing to progressive MS. However, it remains unclear how telomere attrition in MS may be linked to oxidative stress, inflammation and age-related disease processes. Despite few studies in this field, there is substantial evidence on the association of TL and MS. Variability in TL appears to reflect heterogeneity in clinical presentation and course. Further investigations in large and well-characterized cohorts are warranted. More detailed studies on TL of individual chromosomes in specific cell types may help to gain new insights into the pathomechanisms of MS.

Principles and Practical Considerations for the Analysis of Disease-Associated Alternative Splicing Events Using the Gateway Cloning-Based Minigene Vectors pDESTsplice and pSpliceExpress.

Splicing is an important RNA processing step. Genetic variations can alter the splicing process and thereby contribute to the development of various diseases. Alterations of the splicing pattern can be examined by gene expression analyses, by computational tools for predicting the effects of genetic variants on splicing, and by splicing reporter minigene assays for studying alternative splicing events under defined conditions. The minigene assay is based on transient transfection of cells with a vector containing a genomic region of interest cloned between two constitutive exons. Cloning can be accomplished by the use of restriction enzymes or by site-specific recombination using Gateway cloning. The vectors pDESTsplice and pSpliceExpress represent two minigene systems based on Gateway cloning, which are available through the Addgene plasmid repository. In this review, we describe the features of these two splicing reporter minigene systems. Moreover, we provide an overview of studies in which determinants of alternative splicing were investigated by using pDESTsplice or pSpliceExpress. The studies were reviewed with regard to the investigated splicing regulatory events and the experimental strategy to construct and perform a splicing reporter minigene assay. We further elaborate on how analyses on the regulation of RNA splicing offer promising prospects for gaining important insights into disease mechanisms.

Leukocyte Telomere Length in Patients with Multiple Sclerosis and Its Association with Clinical Phenotypes.

Aging is a significant factor influencing the course of multiple sclerosis (MS). Accelerated telomere attrition is an indicator of premature biological aging and a potential contributor to various chronic diseases, including neurological disorders. However, there is currently a lack of studies focusing on telomere lengths in patients with MS. We measured the average leukocyte telomere length (LTL) in biobanked DNA samples of 40 relapsing-remitting MS patients (RRMS), 20 primary progressive MS patients (PPMS), and 60 healthy controls using a multiplex quantitative polymerase chain reaction method. Changes in LTL over a period of >10 years were evaluated in a subset of 10 patients. Association analyses of baseline LTL with the long-term clinical profiles of the patients were performed using inferential statistical tests and regression models adjusted for age and sex. The cross-sectional analysis revealed that the RRMS group was characterized by a significantly shorter relative LTL, on average, as compared to the PPMS group and controls. Shorter telomeres at baseline were also associated with a higher conversion rate from RRMS to secondary progressive MS (SPMS) in the 10-year follow-up. The LTL decrease over time was similar in RRMS patients and PPMS patients in the longitudinal analysis. Our data suggest a possible contributory role of accelerated telomere shortening in the pathobiology of MS. The interplay between disease-related immune system alterations, immunosenescence, and telomere dynamics deserves further investigation. New insights into the mechanisms of disease might be obtained, e.g., by exploring the distribution of telomere lengths in specific blood cell populations.

Systemic Effects by Intrathecal Administration of Triamcinolone Acetonide in Patients With Multiple Sclerosis.

In patients suffering from multiple sclerosis (MS), intrathecal injection of triamcinolone acetonide (TCA) has been shown to improve symptoms of spasticity. Although repeated intrathecal injection of TCA has been used in a number of studies in late-stage MS patients with spinal cord involvement, no clinical-chemical data are available on the distribution of TCA in cerebrospinal fluid (CSF) or serum. Moreover, the effects of intrathecal TCA administration on the concentrations of endogenous steroids remain poorly understood. Therefore, we have quantified TCA and selected endogenous steroids in CSF and serum of TCA-treated MS patients suffering from spasticity. Concentrations of steroids were quantified by LC-MS, ELISA, or ECLIA and compared with the blood-brain barrier status, diagnosed with the Reibergram. The concentration of TCA in CSF significantly increased during each treatment cycle up to >5 µg/ml both in male and female patients (p < 0.001). Repeated TCA administration also evoked serum concentrations of TCA up to >30 ng/ml (p < 0.001) and severely depressed serum levels of cortisol and corticosterone (p < 0.001). In addition, concentrations of circulating estrogen were significantly suppressed (p < 0.001). Due to the potent suppressive effects of TCA on steroid hormone concentrations both in the brain and in the periphery, we recommend careful surveillance of adrenal function following repeated intrathecal TCA injections in MS patients.

Reduced Fragmentation of IGFBP-2 and IGFBP-3 as a Potential Mechanism for Decreased Ratio of IGF-II to IGFBPs in Cerebrospinal Fluid in Response to Repeated Intrathecal Administration of Triamcinolone Acetonide in Patients With Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic autoimmune disease of the brain and spinal cord causing a wide range of symptoms such as impaired walking capability, spasticity, fatigue, and pain. The insulin-like growth factor (IGF) system has regulatory functions for the induction of inflammatory pathways in experimental encephalomyelitis. We have therefore assessed expression and regulation of the IGF system on the level of IGFs and IGFBPs in serum and cerebrospinal fluid (CSF) in the course of four repeated triamcinolone acetonide (TCA) administrations in two female and four male MS patients. Sample series of 20 treatment cycles were analyzed. IGF-I and IGF-II were quantified by ELISAs, and IGFBPs were analyzed by quantitative Western ligand (qWLB) and Western immunoblotting (WIB) in order to differentiate intact and fragmented IGFBPs. The ratios of fragmented to intact IGFBP-2 and -3 were calculated in serum and CSF. Finally, the ratios of IGF-I and IGF-II to the total IGF-binding activity, quantified by qWLB, were determined as an indicator of IGF-related bioactivity. After the fourth TCA administration, the average level of IGF-I was increased in serum (p < 0.001). The increase of IGF-I concentrations in serum resulted in an increased ratio of IGF-I to IGFBPs in the circulation. By contrast in CSF, fragmentation of IGFBP-2 and IGFBP-3 and the ratio of IGF-II to intact IGFBPs were decreased at the fourth TCA administration (p < 0.01). Furthermore, reduced fragmentation of IGFBP-3 in CSF was accompanied by increased concentrations of intact IGFBP-3 (p < 0.001). We conclude that reduced fragmentation of IGFBPs and concomitant reduction of IGF-II to IGFBP ratios indicate regulation of bioactivity of IGF-II in CSF during repeated intrathecal TCA administration in MS patients.

Aberrant expression of alternative splicing variants in multiple sclerosis - A systematic review.

OBJECTIVE: Alternative splicing is an important form of RNA processing that affects nearly all human genes. The differential expression of specific transcript and protein isoforms holds the potential of novel biomarkers for complex diseases. In this systematic review, we compiled the existing literature on aberrant alternative splicing events in multiple sclerosis (MS). METHODS: A systematic literature search in the PubMed database was carried out and supplemented by screening the reference lists of the identified articles. We selected only MS-related original research studies which compared the levels of different isoforms of human protein-coding genes. A narrative synthesis of the research findings was conducted. Additionally, we performed a case-control analysis using high-density transcriptome microarray data to reevaluate the genes that were examined in the reviewed studies. RESULTS: A total of 160 records were screened. Of those, 36 studies from the last two decades were included. Most commonly, peripheral blood samples were analyzed (32 studies), and PCR-based techniques were usually employed (27 studies) for measuring the expression of selected genes. Two studies used an exploratory genome-wide approach. Overall, 27 alternatively spliced genes were investigated. Nine of these genes appeared in at least two studies (CD40, CFLAR, FOXP3, IFNAR2, IL7R, MOG, PTPRC, SP140 and TNFRSF1A). The microarray data analysis confirmed differential alternative pre-mRNA splicing for 19 genes. CONCLUSIONS: An altered RNA processing of genes mediating immune signaling pathways has been repeatedly implicated in MS. The analysis of individual exon-level expression patterns is stimulated by the advancement of transcriptome profiling technologies. In particular, the examination of genes encoded in MS-associated genetic regions may provide important insights into the pathogenesis of the disease and help to identify new biomarkers.

The Cerebrospinal Fluid in Multiple Sclerosis.

Investigation of cerebrospinal fluid (CSF) in the diagnostic work-up in suspected multiple sclerosis (MS) patients has regained attention in the latest version of the diagnostic criteria due to its good diagnostic accuracy and increasing issues with misdiagnosis of MS based on over interpretation of neuroimaging results. The hallmark of MS-specific changes in CSF is the detection of oligoclonal bands (OCB) which occur in the vast majority of MS patients. Lack of OCB has a very high negative predictive value indicating a red flag during the diagnostic work-up, and alternative diagnoses should be considered in such patients. Additional molecules of CSF can help to support the diagnosis of MS, improve the differential diagnosis of MS subtypes and predict the course of the disease, thus selecting the optimal therapy for each patient.

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.

Impact of hyperglycemia on autoimmune pancreatitis and regulatory T-cells.

AIM: To evaluate the influence of hyperglycemia on the progression of autoimmune pancreatitis. METHODS: We induced hyperglycemia by repetitive intraperitoneal (ip) injection of 50 mg/kg streptozotocin in MRL/MpJ mice, which develop autoimmune pancreatitis due to a genetic predisposition. We compared the extent of inflammation (histological score, CD3+ lymphocytes, CD8+ T-cells, CD4+ T-cells, Foxp3+ T-helper cells) in the pancreas of hyperglycemic and normoglycemic mice. We also analyzed the number of leukocytes, lymphocytes, granulocytes and monocytes in the blood. In addition, we determined the percentage of CD3+ lymphocytes, CD8+ T-cells, CD4+ T-cells, Foxp3+ T-helper cells, Foxp3+ CD25+ T-helper and Foxp3- T-helper cells in the spleen by flow cytometry. RESULTS: Treatment with streptozotocin caused a strong induction of hyperglycemia and a reduction in body weight (P < 0.001). Severe hyperglycemia did not, however, lead to an aggravation, but rather to a slight attenuation of autoimmune pancreatitis. In the pancreas, both the histological score of the pancreas as well as the number of CD3+ lymphocytes (P < 0.053) were decreased by hyperglycemia. No major changes in the percentage of CD8+ T-cells, CD4+ T-cells, Foxp3+ T-helper cells were observed between hyperglycemic and normoglycemic mice. Hyperglycemia increased the numbers of leukocytes (P < 0.001), lymphocytes (P = 0.016), granulocytes and monocytes (P = 0.001) in the blood. Hyperglycemia also moderately reduced the percentage of CD3+ lymphocytes (P = 0.057), significantly increased the percentage of Foxp3+ T-helper cells (P = 0.018) and Foxp3+ CD25+ T-helper cells (P = 0.021) and reduced the percentage of Foxp3- T-helper cells (P = 0.034) in the spleen. CONCLUSION: Hyperglycemia does not aggravate but moderately attenuates autoimmune pancreatitis, possibly by increasing the percentage of regulatory T-cells in the spleen.

Microarray data of transcriptome shifts in blood cell subsets during S1P receptor modulator therapy.

Treatment with fingolimod, a sphingosine-1-phosphate (S1P) receptor modulator, prevents the egress of immune cell subpopulations from lymphoid tissues into the blood. We obtained peripheral blood samples from patients with relapsing multiple sclerosis before the initiation of fingolimod therapy, after one day and after 3 months. To investigate the differential expression induced by the drug, five different cell populations were isolated. We then employed 150 Human Transcriptome Arrays (HTA 2.0) interrogating >245,000 protein-coding and >40,000 non-coding transcript isoforms. After 3 months of treatment, CD4+ and CD8+ T-cells showed huge transcriptome shifts, whereas the profiles of B-cells (CD19+) were slightly altered and those of monocytes (CD14+) and natural killer cells (CD56+) remained unaffected. With >6 million probes for exons and splice junctions, our large HTA 2.0 dataset provides a deep view into alternative splicing patterns in immune cell subsets. Our data may also be useful for comparing the effects on gene expression signatures of novel S1P receptor modulators, which are currently tested in clinical trials for other autoimmune and neurodegenerative 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.

Fingolimod alters the transcriptome profile of circulating CD4+ cells in multiple sclerosis.

Multiple sclerosis is a demyelinating disease affecting the central nervous system. T cells are known to contribute to this immune-mediated condition. Fingolimod modulates sphingosine-1-phosphate receptors, thereby preventing the egress of lymphocytes, especially CCR7-expressing CD8+ and CD4+ T cells, from lymphoid tissues. Using Affymetrix Human Transcriptome Arrays (HTA 2.0), we performed a transcriptome profiling analysis of CD4+ cells obtained from the peripheral blood of patients with highly active relapsing-remitting multiple sclerosis. The samples were drawn before the first administration of fingolimod as well as 24 hours and 3 months after the start of therapy. Three months after treatment initiation, 890 genes were found to be differentially expressed with fold-change >2.0 and t-test p-value < 0.001, among them several microRNA precursors. A subset of 272 genes were expressed at lower levels, including CCR7 as expected, while 618 genes showed an increase in expression, e.g., CCR2, CX3CR1, CD39, CD58 as well as LYN, PAK1 and TLR2. To conclude, we studied the gene expression of CD4+ cells to evaluate the effects of fingolimod treatment, and we identified 890 genes to be altered in expression after continuous drug administration. T helper cells circulating in the blood during fingolimod therapy present a distinct gene expression signature.

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.

High-Density Peptide Microarray Analysis of IgG Autoantibody Reactivities in Serum and Cerebrospinal Fluid of Multiple Sclerosis Patients.

Intrathecal immunoglobulin G (IgG) synthesis and oligoclonal IgG bands in cerebrospinal fluid (CSF) are hallmarks of multiple sclerosis (MS), but the antigen specificities remain enigmatic. Our study is the first investigating the autoantibody repertoire in paired serum and CSF samples from patients with relapsing-remitting MS (RRMS), primary progressive MS (PPMS), and other neurological diseases by the use of high-density peptide microarrays. Protein sequences of 45 presumed MS autoantigens (e.g.MOG, MBP, and MAG) were represented on the microarrays by overlapping 15mer peptides. IgG reactivities were screened against a total of 3991 peptides, including also selected viral epitopes. The measured antibody reactivities were highly individual but correlated for matched serum and CSF samples. We found 54 peptides to be recognized significantly more often by serum or CSF antibodies from MS patients compared with controls (pvalues <0.05). The results for RRMS and PPMS clearly overlapped. However, PPMS patients presented a broader peptide-antibody signature. The highest signals were detected for a peptide mapping to a region of the Epstein-Barr virus protein EBNA1 (amino acids 392-411), which is homologous to the N-terminal part of human crystallin alpha-B. Our data confirmed several known MS-associated antigens and epitopes, and they delivered additional potential linear epitopes, which await further validation. The peripheral and intrathecal humoral immune response in MS is polyspecific and includes antibodies that are also found in serum of patients with other diseases. Further studies are required to assess the pathogenic relevance of autoreactive and anti-EBNA1 antibodies as well as their combinatorial value as biomarkers for MS.

Molecular biomarkers in cerebrospinal fluid of multiple sclerosis patients.

Multiple sclerosis (MS) is a chronic immune-mediated disease of the central nervous system, usually occurring in young adults and leading to disability. Despite the progress in technology and intensive research work of the last years, diagnosing MS can still be challenging. A heterogenic and complex pathophysiology with various types of disease courses makes MS unique for each patient. There is an urgent need to identify markers facilitating rapid and accurate diagnosis and prognostic assessments with regard to optimal therapy for each MS patient. Cerebrospinal fluid (CSF) is an outstanding source of specific markers related to MS pathology. Molecules reflecting specific pathological processes, such as inflammation, cellular damage, and loss of blood-brain-barrier integrity, are detectable in CSF. Clinically used biomarkers of CSF are oligoclonal bands, IgG-index, measles-rubella-zoster-reaction, anti-aquaporin 4 antibodies, and antibodies against John Cunningham virus. Many other potential biomarkers have been proposed in recent years. In this review we examine the current scientific knowledge on CSF molecular markers that could guide diagnosis and discrimination of different MS forms, support treatment decisions, or be helpful in monitoring and predicting disease progression, therapy response, and complications such as opportunistic infections.