Search

CD4+ T cells are considered the main orchestrators of autoimmune diseases. Their disruptive effect on CD4+ T cell differentiation and the imbalance between T helper cell populations can be most accurately determined using experimental autoimmune encephalomyelitis (EAE) as an animal model of multiple sclerosis (MS). One epigenetic factor known to promote autoimmune inflammation is miRNA-155 (miR-155), which is significantly upregulated in inflammatory T cells. The aim of the present study was to profile the transcriptome of immunized mice and determine their gene expression levels based on mRNA and miRNA sequencing. No statistically significant differences in miRNA profile were observed; however, substantial changes in gene expression between miRNA-155 knockout (KO) mice and WT were noted. In miR-155 KO mice, mRNA expression in CD4+ T cells changed in response to immunization with the myeloid antigen MOG 35-55 . After restimulation with MOG 35-55 , increased Ffar1 (free fatty acid receptor 1) and Scg2 (secretogranin-2) expression were noted in the CD4+ T cells of miR-155-deficient mice; this is an example of an alternative response to antigen stimulation.

Almost all currently licensed disease-modifying therapies (DMTs) for MS treatment require prolonged if not lifelong administration. Yet, as people age, the immune system has increasingly reduced responsiveness, known as immunosenescence. Many MS DMTs reduce the responsiveness of the immune system, increasing the risks for infections and possibly cancers. As people with MS (pwMS) age, it is recognized that inflammatory MS activity declines. Several studies have addressed de-escalation of DMTs for relapsing MS under special circumstances. Here, we review evidence for de-escalating DMTs as a strategy that is particularly relevant to pwMS of older age. Treatment de-escalation can involve various strategies, such as extended or reduced dosing, switching from high-efficacy DMTs having higher risks to moderately effective DMTs with lesser risks, or treatment discontinuation. Studies have suggested that for natalizumab extended dosing maintained clinical efficacy while reducing the risk of PML. Extended interval dosing of ocrelizumab mitigated the decline of Ig levels. Retrospective and observational discontinuation studies demonstrate that age is an essential modifier of drug efficacy. Discontinuation of MS treatment in older patients has been associated with a stable disease course, while younger patients who discontinued treatment were more likely to experience new clinical activity. A recently completed 2-year randomized-controlled discontinuation study in 260 stable pwMS > 55 years found stable clinical multiple sclerosis with only a small increased risk of new MRI activity upon discontinuation. DMT de-escalation or discontinuation in MS patients older than 55 years may be non-inferior to continued treatment with immunosuppressive agents having higher health risks. However, despite several small studies, a definite conclusion about treatment de-escalation in older pwMS will require larger and longer studies. Ideally, comparison of de-escalation versus continuation versus discontinuation of DMTs should be done by prospective randomized-controlled trials enrolling sufficient numbers of subjects to allow comparisons for MS patients of both sexes within age groups, such as 55-59, 60-65, 66-69, etc. Optimally, such studies should be 3 years or longer and should incorporate testing for specific markers of immunosenescence (such as T-cell receptor excision circles) to account for differential aging of individuals., (© 2024. The Author(s).)

Background and Objectives: X-linked adrenomyeloneuropathy (AMN) is an inherited neurodegenerative disorder associated with mutations in the ABCD1 gene and the accumulation of very long-chain fatty acids (VLFCAs) in plasma and tissues. Currently, there is no effective treatment for AMN. We have aimed to evaluate the therapeutic effects of mesenchymal stem cell (MSC) transplantation in patients with AMN., Methods: This is a small cohort open-label study with patients with AMN diagnosed and treated at the University Hospital in Olsztyn, Poland. All patients met clinical, biochemical, MRI, and neuropsychological criteria for AMN. MSCs derived from Wharton jelly, 20 × 10 6 cells, were administered intrathecally three times every 2 months, and patients were followed up for an additional 3 months. The primary outcome measures included a blinded assessment of lower limb muscle strength with the Medical Research Council Manual Muscle Testing scale at baseline and on every month visits until the end of the study. Additional outcomes included measurements of the timed 25-feet walk (T25FW) and VLFCA serum ratio., Results: Three male patients with AMN with an age range of 26-37 years participated in this study. All patients experienced increased muscle strength in the lower limbs at the end of the study versus baseline. The power grade increased by 25-43% at the baseline. In addition, all patients showed an improvement trend in walking speed measured with the T25FW test. Treatment with MSCs in patients with AMN appeared to be safe and well tolerated., Discussion: The results of this study demonstrated that intrathecal administration of WJ-MSC improves motor symptoms in patients with AMN. The current findings lend support to the safety and feasibility of MSC therapy as a potentially viable treatment option for patients with AMN., Competing Interests: KS has received personal compensation for consulting from Biogen, Celgene, GeNeuro, Merck, Novartis, Polpharma, Sanofi, Roche, TG Therapeutics, and received research support from Merck and Roche. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Siwek, Zwiernik, Jezierska-Woźniak, Jezierska, Mycko and Selmaj.)

Multiple sclerosis is a chronic demyelinating disease with different disease phenotypes. The current FDA-approved disease-modifying therapeutics (DMTs) cannot cure the disease, but only alleviate the disease progression. While the majority of patients respond well to treatment, some of them are suffering from rapid progression. Current drug delivery strategies include the oral, intravenous, subdermal, and intramuscular routes, so these drugs are delivered systemically, which is appropriate when the therapeutic targets are peripheral. However, the potential benefits may be diminished when these targets sequester behind the barriers of the central nervous system. Moreover, systemic drug administration is plagued with adverse effects, sometimes severe. In this context, it is prudent to consider other drug delivery strategies improving their accumulation in the brain, thus providing better prospects for patients with rapidly progressing disease course. These targeted drug delivery strategies may also reduce the severity of systemic adverse effects. Here, we discuss the possibilities and indications for reconsideration of drug delivery routes (especially for those "non-responding" patients) and the search for alternative drug delivery strategies. More targeted drug delivery strategies sometimes require quite invasive procedures, but the potential therapeutic benefits and reduction of adverse effects could outweigh the risks. We characterized the major FDA-approved DMTs focusing on their therapeutic mechanism and the potential benefits of improving the accumulation of these drugs in the brain., (© 2023 Wiley Periodicals LLC.)

Environmental triggers often work via signal transduction cascades that modulate the epigenome and transcriptome of cell types involved in the disease process. Multiple sclerosis (MS) is an autoimmune disease affecting the central nervous system being characterized by a combination of recurring inflammation, demyelination and progressive loss of axons. The mechanisms of MS onset are not fully understood and genetic variants may explain only some 20% of the disease susceptibility. From the environmental factors being involved in disease development low vitamin D levels have been shown to significantly contribute to MS susceptibility. The pro-hormone vitamin D 3 acts via its metabolite 1α,25-dihydroxyvitamin D 3 (1,25(OH) 2 D 3 ) as a high affinity ligand to the transcription factor VDR (vitamin D receptor) and is a potent modulator of the epigenome at thousands of genomic regions and the transcriptome of hundreds of genes. A major target tissue of the effects of 1,25(OH) 2 D 3 and VDR are cells of innate and adaptive immunity, such as monocytes, dendritic cells as well as B and T cells. Vitamin D induces immunological tolerance in T cells and reduces inflammatory reactions of various types of immune cells, all of which are implicated in MS pathogenesis. The immunomodulatory effects of 1,25(OH) 2 D 3 contribute to the prevention of MS. However, the strength of the responses to vitamin D 3 supplementation is highly variegated between individuals. This review will relate mechanisms of individual's vitamin D responsiveness to MS susceptibility and discuss the prospect of vitamin D 3 supplementation as a way to extinguish the autoimmunity in MS.

Circular RNA (circRNA) molecules represent a novel and unique class of endogenous non-coding RNAs controlling the expression and function of microRNA (miRNA) and post-transcriptional regulation. Recent studies implicated circRNA in the pathomechanism of multiple sclerosis (MS). Hybridization microarray was used to define the circRNA profile in the peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with relapsing-remitting MS (RRMS: 10 in relapse, 10 in remission) and 10 healthy controls (HCs). We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) with an independent cohort of 45 RRMS patients (18 in relapse, 27 in remission) and 27 HCs. Microarray analysis revealed 246 circRNAs differentially downregulated ( P  < 0.05) in RRMS patients versus HCs. We validated two circRNAs of the three showing the lowest levels of differential expression in the RRMS remission group versus the HC group: hsa&#95;circRNA&#95;101145 and hsa&#95;circRNA&#95;001896. Their expression was significantly decreased during remission in RRMS ( P  = 0.0000332, FC = 0.385 and P  = 0.0455, FC = 0.591, respectively) and in patients showing a lower level of disability (hsa&#95;circRNA&#95;101145, P  = 0.0695; hsa&#95;circRNA&#95;001896, P  = 0.0008). Bioinformatic analysis revealed 10 miRNAs interacting with these circRNAs in a complementary manner and led to the discovery of three protein-coding mRNAs downregulated in patients with RRMS during remission. These transcripts have been previously implicated in oxidative stress, blood-brain barrier permeability, microglia function, and extracellular matrix molecules altering the microenvironment and inhibiting oligodendrocyte progenitor cells. circRNAs displayed a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNAs with low expression in important mechanistic pathways of RRMS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Mycko, Zurawska, Selmaj and Selmaj.)

Purpose of Review: Multiple sclerosis (MS) is highly heterogenic disorder with respect to clinical course, diagnosis, and treatment response. There is an urgent need to search for simply and reliable fluid body biomarker which would assist the diagnosis and prediction of clinical and treatment prognosis., Recent Findings: 'Traditional' MS biomarkers, with exception of cerebrospinal fluid oligoclonal bands, still are having limited clinical value. Therefore, there is growing interest in novel molecules and ingredients. The most robust results have been generated with regard to cerebrospinal fluid and serum levels of neurofilament light chains (NfL). However, there are still some limitations related to specificity of NfL which delays its use in everyday practice. We present a new approach to search for biomarkers involving extracellular RNA, particularly microRNA (miRNA), and small extracellular vesicles. MiRNA represents an important molecular mechanism influencing gene expression, including those involved in MS pathogenesis and extracellular vesicles transfer multiple cargo, including myelin molecules from parental cells of central nervous system to the long-distance targets., Summary: MiRNAs which control gene expression in cells involved in autoimmune processes in MS as well as extracellular vesicles transferring myelin content might generate a new promising categories of biomarkers of MS., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background and Objectives: To investigate the total circular RNA (circRNA) profile in patients with relapsing-remitting multiple sclerosis (RRMS) and healthy controls (HCs)., Methods: Hybridization microarray was used to define the circRNA profile in peripheral blood mononuclear cells (PBMCs) from 20 untreated patients with RRMS (10 in relapse and 10 in remission) and 10 HCs. We analyzed close to 14,000 individual circRNAs per sample. The discovery set data were validated using quantitative reverse transcription-PCR with an independent cohort of 47 patients with RRMS (19 in relapse and 28 in remission) and 27 HCs., Results: Microarray analysis revealed 914 transcripts to be differentially expressed between patients with RRMS in relapse and HCs ( p < 0.05). We validated 3 circRNAs from 5 showing highest levels of differential expression in the RRMS relapse vs HC group: hsa&#95;circRNA&#95;101348, hsa&#95;circRNA&#95;102611, and hsa&#95;circRNA&#95;104361. Their expression was significantly increased during relapse in RRMS ( p = 0.0002, FC = 2.9; p = 0.01, FC = 1.6; and p = 0.001, FC = 1.5, respectively) and in patients showing gadolinium enhancement on brain MRI (hsa&#95;circRNA&#95;101348, p = 0.0039, FC = 2.4; hsa&#95;circRNA&#95;104361, p = 0.029, FC = 1.7). Bioinformatic analysis revealed 15 microRNAs interacting with these circRNAs in a complementary manner and led to the discovery and validation of 3 protein-coding RNAs upregulated in patients with RRMS during relapse. Two of these, AK2 and IKZF3 , have previously been implicated in B-cell function., Discussion: circRNAs display a distinct profile in PBMCs from patients with RRMS, and our results may implicate circRNA in the known disturbed B-cell activity in RRMS and thus represent a novel biomarker for monitoring relapse activity., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

T helper cells type 17 (Th17) are orchestrators of autoimmune conditions, including multiple sclerosis (MS), but mechanisms of Th17 pathogenicity remain unknown. MicroRNAs (miRNA) are known to control T cells. To understand the function of miRNA in Th17, we have established a T cell line, EL4-TCR + , that resembles the expression pattern of the Th17 cells. Subsequently, we have evaluated the crosstalk between miRNA and Th17 genes' expression using a combination of gene expression profiling, gene expression manipulation, RNA and protein immunoprecipitation, as well as confocal microscopy. We have found that Th17-related miRNA were strongly expressed in EL4-TCR + cells following the binding of the cluster of differentiation 3 (CD3) component of the T cell receptor (TCR). Furthermore, a specific inhibition of these miRNA resulted in downregulation of the critical Th17 genes' expression. Surprisingly, this mechanism relied on the function of the stress signal regulator heat shock protein 70 (HSP70). Upon activation, HSP70 co-localized intracellularly with miRNA processing proteins. Precipitation of HSP70 resulted in enrichment of the Th17-associated miRNA. Finally, HSP70 inhibition led to downregulation of the Th17 genes' expression and ameliorated development of autoimmune demyelination. Our study demonstrated that HSP70 facilitates specific miRNA function leading to Th17 genes' expression, a mechanism linking stress and autoimmunity.

New DNA sequencing technologies have uncovered non-coding RNA (ncRNA) as a major player in regulating cellular processes and can no longer be dismissed as "junk" or "dark" RNA. Among the ncRNA, microRNA (miRNA) is arguably the most extensively characterized category and a number of studies have implicated them in regulating critical functions that can influence autoimmune demyelination. Of specific interest to multiple sclerosis (MS), miRNA have been implicated in both regulating immune responses and myelination, thus making them an attractive candidate for both pharmacological intervention and as disease biomarkers. In addition, exosomes, small vesicles secreted by most cell types and present in all body fluids, have been also shown to play roles in immune signaling, inflammation and angiogenesis. Therefore, exosomes are also being explored as tools for therapeutic delivery and as biomarkers. This article reviews the recent advances in miRNA and exosome profiling in MS and experimental models.

Introduction: Research into the mechanisms of autoimmune demyelination have highlighted B cells in this process. Therapies targeting this population were a recent addition to the multiple sclerosis (MS) drugs portfolio. The SARS-CoV-2 pandemic and the risk of severe COVID-19 have challenged the safety of B cell depletion in MS patients., State of the Art: Selective depletion of B cells by monoclonal antibodies as monotherapy in MS has been shown to profoundly suppress disease activity among relapsing-remitting MS patients. Furthermore ocrelizumab, a humanised anti-CD20 monoclonal antibody, was the first licensed therapy in primary progressive MS. Based on the concept of the role of B cells in MS, many therapeutic approaches are emerging as novel ways to treat autoimmune demyelination. However, during the SARS-CoV-2 pandemic, a conservative approach toward limiting immune suppression in MS patients has been proposed., Clinical Implications: Emerging evidence does not support the notion of increased susceptibility among MS patients to the SARS-CoV-2 infection, or any predisposition toward greater severity of COVID-19. This also does not appear to be the case for MS patients undergoing B cell depletion therapies. Thus, any decision to withhold immune suppression in MS patients during the SARS-CoV-2 pandemic is probably incorrect. MS therapeutic decision-making should focus on the danger of poorly controlled autoimmune demyelination rather than perceived risks from COVID-19., Future Directions: The current pandemic highlights the need to develop more selective and safer methods of immunomodulation in MS. B cells represent several functionally different populations. Further research into the different role of these cells during autoimmune demyelination should yield better, safer strategies to control the encephalitogenic process.

Multiple sclerosis (MS) is believed to be an autoimmune disease of the central nervous system (CNS) in which autoreactive immune cells recognizing myelin antigens lead to demyelination and axonal injury. Mechanisms inducing and controlling the pathogenesis of MS have not been fully elucidated. Recent studies suggest an important role of epigenetic processes during the development of MS. One of the most significant discoveries in the field of epigenetic contribution to immune response has been the recognition of a group of microRNAs (miRNAs). These single-stranded non-coding RNA molecules regulate the expression of genes encoding proteins and have already been shown to be involved in pathogenesis of MS. Some miRNAs enhance generation of pro-inflammatory immune cells by promoting Th1 and Th17 pathways and others contribute to regulatory and tissue repair processes. The miRNA-dependent controlling process of autoimmune reactions is highly complex because of miRNA redundancy and multitarget nature of most of these molecules. Recently it was discovered that circular RNAs (circRNA) representing a new class of RNA possess a unique ability to control miRNAs by blocking their activity. CircRNAs are called natural miRNA "sponges" as the single circRNA molecule is able to neutralize several miRNAs and thus might determine the availability of miRNAs for their posttranscription regulation. Thus, circRNAs emerged as critical factors in epigenetic regulation of many human diseases including MS. In addition, in contrary to other RNA species they are very stable in the blood and other biological fluids and thus might be considered as a candidate for a biomarker of MS., (Copyright © 2019. Published by Elsevier B.V.)

Background: Exosomes are small extracellular vesicles that provide cell-to-cell communication and are involved in immunoregulation., Objective: To investigate serum exosomes for the presence of myelin proteins outside the central nervous system (CNS) and their role in multiple sclerosis (MS)., Methods: Serum, cerebrospinal fluid (CSF), and peripheral blood mononuclear cell (PBMC) samples were collected from 45 patients with relapsing-remitting MS (RRMS), 30 patients with secondary progressive MS (SPMS), and 45 healthy controls. Exosomes were isolated using a polymer formulation method, and their size, concentration, and CNS myelin protein contents were measured by a nanoparticle tracking analysis, enzyme-linked immunosorbent assays, and Western blot., Results: We found that exosomes expressed three major myelin proteins, myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein (MOG). Exosomal content of MOG strongly correlated with disease activity and was highest in RRMS patients in relapse and in SPMS patients. Serum-derived exosomes induced proliferation of MOG-T cell receptor transgenic T cells confirming that serum exosomes maintained MOG immunogenicity., Conclusion: Exosomes isolated outside CNS tissue expressed myelin proteins, and the presence of MOG correlated strongly with disease activity. We conclude that exosomes might enhance and/or perpetuate anti-myelin immune reactions in MS and may provide novel markers of disease activity.

Multiple sclerosis (MS) is a putative autoimmune disease of the central nervous system (CNS) in which autoreactive immune cells recognizing myelin antigens lead to demyelination and axonal injury. Mechanisms relevant to the pathogenesis of MS have not been fully elucidated, particularly those underlying initiation of immune system dysfunction. For example, it is not known how reactivity against CNS components is generated within the peripheral immune system. In this review, we propose that a significant contribution to the immunoregulatory events may derive from a cell-to-cell communication system involving the production, secretion and transfer of extracellular vesicles known as exosomes. Herein, we discuss in detail the biogenesis and roles of these cell surface-generated vesicles from the standpoint of receptors and their cargo, microRNA. It is well known that exosomes can cross the blood-brain barrier and thus may contribute to the spread of brain antigens to the periphery. Further understanding of exosome-dependent mechanisms in MS should provide a novel angle to the analysis of the pathogenesis of this disease. Finally, we launch the idea that exosomes and their contents may serve as biomarkers in MS., (Copyright © 2017. Published by Elsevier B.V.)

Objective: Accumulating evidence supports a role for exosomes in immune regulation. In this study, we investigated the total circulating exosome transcriptome in relapsing-remitting multiple sclerosis (RRMS) patients and healthy controls (HC)., Methods: Next generation sequencing (NGS) was used to define the global RNA profile of serum exosomes in 19 RRMS patients (9 in relapse, 10 in remission) and 10 HC. We analyzed 5 million reads and >50,000 transcripts per sample, including a detailed analysis of microRNAs (miRNAs) differentially expressed in RRMS. The discovery set data were validated by quantification using digital quantitative polymerase chain reaction with an independent cohort of 63 RRMS patients (33 in relapse, 30 in remission) and 32 HC., Results: Exosomal RNA NGS revealed that of 15 different classes of transcripts detected, 4 circulating exosomal sequences within the miRNA category were differentially expressed in RRMS patients versus HC: hsa-miR-122-5p, hsa-miR-196b-5p, hsa-miR-301a-3p, and hsa-miR-532-5p. Serum exosomal expression of these miRNAs was significantly decreased during relapse in RRMS. These miRNAs were also decreased in patients with a gadolinium enhancement on brain magnetic resonance imaging. In vitro secretion of these miRNAs by peripheral blood mononuclear cells was also significantly impaired in RRMS., Interpretation: These data show that circulating exosomes have a distinct RNA profile in RRMS. Because putative targets for these miRNAs include the signal transducer and activator of transcription 3 and the cell cycle regulator aryl hydrocarbon receptor, the data suggest a disturbed cell-to-cell communication in this disease. Thus, exosomal miRNAs might represent a useful biomarker to distinguish multiple sclerosis relapse. Ann Neurol 2017;81:703-717., (© 2017 American Neurological Association.)

Recent evidence has revealed an unsuspected suppressive role played by neutrophils during microbial infections. An especially intriguing aspect of this role is the ability of neutrophils to produce interleukin (IL)-10 following interaction with lipopolysaccharide (LPS)-stimulated regulatory T (Treg) cells. The present study demonstrates that generation of IL-10 in neutrophils induced by LPS-stimulated Treg cells required direct cell-cell contact. This effect was dependent on the binding of CD11b and intercellular adhesion molecule 1. Neither stimulation of neutrophils with LPS nor their culture with unstimulated Treg cells, CD3/CD28 monoclonal antibodies-stimulated Treg cells, or T conventional cells affected intracellular IL-10 expression. IL-10-positive neutrophils were also induced by exogenous IL-10, providing an example of a positive feedback loop. Both LPS-stimulated Treg cells and exogenous IL-10 exclusively promoted posttranslational modifications of histones, H3K4me3 and H3Ac Lys4, that activate IL-10 genomic locus in neutrophils, while the promoter of IL-10 gene was inactive in resting, LPS-stimulated neutrophils, following blocking of direct interaction with LPS-stimulated Treg cells or in LPS-preactivated neutrophils incubated with LPS-stimulated Treg cells. We additionally confirmed the presence of IL-10-producing neutrophils in vivo in patients with periodontal abscess induced by Gram-negative bacteria, as opposed to neutrophils isolated from the site of aseptic inflammation in patients with neuromyelitis optica.

microRNA-155 (miR-155) plays an important role in posttranscriptional gene regulation of the immune system. We and others have described miR-155 upregulation in T helper cells (Th) during the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. We have shown that mice in which the miR-155 host gene (MIR155HG) has been deactivated are resistant to EAE. MIR155HG produces two different miRNA strands, miR-155-5p and miR-155-3p, and miR-155-5p has been considered the only functional miR-155 form. Surprisingly, we found that miR-155-3p is also strongly upregulated in Th cells infiltrating the brain in EAE. Functional manipulation of miR-155-3p expression revealed its particular role in regulation of Th17 development. The search for miRNA-155-3p target genes highlighted transcripts of two heat shock protein 40 genes, Dnaja2 and Dnajb1. These two genes negatively regulated Th17 differentiation, leading to decreased EAE. Therefore, our findings provide new insights into a previously unknown mechanism by which miR-155-3p controls Th17 cell differentiation and autoimmune demyelination., Significance Statement: Multiple sclerosis (MS) is brain-specific autoimmune disease mediated by T helper (Th) cells autoreactive to myelin. The mechanisms leading to MS are not fully understood and microRNAs (miRNAs) emerge as important regulators of the process. We report that, in an MS murine model of experimental autoimmune encephalomyelitis, miR-155 controls Th cell function by an unusual mechanism involving a rare form, miR-155-3p. miR-155-3p is specifically found in brain-infiltrating myelin-autoreactive CD4(+) T cells and contributes to the development of an encephalitogenic Th17 population. miR-155-3p promotes Th17 by inhibiting two heat shock protein 40 genes, Dnaja2 and Dnajb1. Our findings indicate a unique miRNA function in the brain-infiltrating Th cells and suggest Dnaja2 and Dnajb1 as targets for intervention in autoimmune demyelination., (Copyright © 2015 the authors 0270-6474/15/3516504-12$15.00/0.)

Notch receptors (Notch1-4) are involved in the differentiation of CD4 T cells and the development of autoimmunity. Mechanisms regulating Notch signaling in CD4 T cells are not fully elucidated. In this study we investigated potential crosstalk between Notch pathway molecules and heat shock protein 70 (Hsp70), the major intracellular chaperone involved in the protein transport during immune responses and other stress conditions. Using Hsp70(-/-) mice we found that Hsp70 is critical for up-regulation of NICD1 and induction of Notch target genes in Jagged1- and Delta-like1-stimulated CD4 T cells. Co-immunoprecipitation analysis of wild-type CD4 T cells stimulated with either Jagged1 or Delta-like1 showed a direct interaction between NICD1 and Hsp70. Both molecules co-localized within the nucleus of CD4 T cells stimulated with Notch ligands. Molecular interaction and nuclear colocalization of NICD1 and Hsp70 were also detected in CD4 T cells reactive against myelin oligodendrocyte glycoprotein (MOG)35-55, which showed Hsp70-dependent up-regulation of both NICD1 and Notch target genes. In conclusion, we demonstrate for the first time that Hsp70 interacts with NICD1 and contributes to the activity of Notch signaling in CD4 T cells. Interaction between Hsp70 and NICD1 may represent a novel mechanism regulating Notch signaling in activated CD4 T cells., (Copyright © 2015 Elsevier B.V. All rights reserved.)

MicroRNAs (miRNAs) associate with Argonaute (Ago), GW182, and FXR1 proteins to form RNA-induced silencing complexes (RISCs). RISCs represent a critical checkpoint in the regulation and bioavailability of miRNAs. Recent studies have revealed dysregulation of miRNAs in multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE); however, the function of RISCs in EAE and MS is largely unknown. Here, we examined the expression of Ago, GW182, and FXR1 in CNS tissue, oligodendrocytes (OLs), brain-infiltrating T lymphocytes, and CD3(+)splenocytes (SCs) of EAE mic, and found that global RISC protein levels were significantly dysregulated. Specifically, Ago2 and FXR1 levels were decreased in OLs and brain-infiltrating T cells in EAE mice. Accordingly, assembly of Ago2/GW182/FXR1 complexes in EAE brain tissues was disrupted, as confirmed by immunoprecipitation experiments. In parallel with alterations in RISC complex content in OLs, we found downregulation of miRNAs essential for differentiation and survival of OLs and myelin synthesis. In brain-infiltrating T lymphocytes, aberrant RISC formation contributed to miRNA-dependent proinflammatory helper T-cell polarization. In CD3(+) SCs, we found increased expression of both Ago2 and FXR1 in EAE compared with nonimmunized mice. Therefore, our results demonstrate a gradient in expression of miRNA between primary activated T cells in the periphery and polarized CNS-infiltrating T cells. These results suggest that, in polarized autoreactive effector T cells, miRNA synthesis is inhibited in response to dysregulated RISC assembly, allowing these cells to maintain a highly specific proinflammatory program. Therefore, our findings may provide a mechanism that leads to miRNA dysregulation in EAE/MS., (Copyright © 2015 the authors 0270-6474/15/357521-17$15.00/0.)

Plasmacytoid dendritic cells (pDCs), an important immunoregulatory population, are characterized by vigorous secretion of type I interferons (IFNs) in response to toll-like receptor (TLR) 7 and 9 stimulation. We studied the function of pDCs in multiple sclerosis (MS) patients by analysis of TLR7 responses. We assessed a pDC secretion pattern of cytokines in the short term PBMC cultures stimulated with TLR7 agonist. pDCs sorted from PBMCs of both MS patients and controls were used to assess TLR7 expression profile. TLR7 induced signaling in pDCs has been analyzed with intracellular flow cytometry. We have identified a clinically correlated significant decrease of the TLR7-induced IFN-alfa (IFNa) secretion by pDCs from MS patients. This deficit has been accompanied by insufficient intracellular phosphorylation of protein kinase Akt and a decrease of the TLR7 gene expression in MS pDCs. Our results demonstrated a selective pDC deficit in MS supporting a relationship between pDCs and mechanisms of MS., (Copyright © 2014 Elsevier Inc. All rights reserved.)

The CNS is considered an immune privileged site because its repertoire of highly immunogenic molecules remains unseen by the immune system under normal conditions. However, the mechanism underlying the inhibition of immune reactions within the CNS environment is not known, particularly in regions containing myelin, which contains several potent proteins and lipids that are invariably recognized as foreign by immune system cells. Sulfatides constitute a major component of myelin glycolipids and are known to be capable of raising an immune response. In this study, the effect of sulfatides on mouse T cell function and differentiation was analyzed in vitro and in vivo. We found profound inhibition of sulfatide-dependent T cell proliferation which was particularly pronounced in naive T helper (Th) cells. The inhibitory effect of sulfatides on T cell function was CD1d-independent and was not related to apoptosis or necrosis but did involve the induction of anergy as confirmed by the upregulation of early growth response 2 transcription factor. A glycolipid 3-sulfate group was essential for the T cell suppression, and the T cell inhibition was galectin-4-dependent. Sulfatide stimulation in vitro led to prominent suppression of Th17 differentiation, and this was related to a decrease in susceptibility to disease in a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis. Thus, we have defined a novel mechanism of negative regulation of T cell function by endogenous brain-derived glycolipids, a family of molecules traditionally deemphasized in favor of myelin proteins in studies of CNS autoimmunity., (Copyright © 2014 the authors 0270-6474/14/348646-13$15.00/0.)

This study tested the hypothesis that CD4(+)CD25(+)CD127(low) regulatory T (Treg) cells might induce immunosuppressive properties in apoptotic neutrophils. Treg cells are recognized as a major subset of immune cells possessing potent suppressive properties directed at T effector cells. However, Treg cells have recently been found to inhibit neutrophil function and promote their apoptosis. One of the mechanisms of action of Treg cells is the induction of other suppressor cell populations according to an infectious tolerance model. We showed that LPS-activated Treg cells promote generation of IL-10 and TGF-β1, inhibit IL-6 production by PMNs and induce the expression of heme oxygenase-1 (HO-1) and the suppressor of cytokine signaling 3 molecule (SOCS3). However, CD3/CD28-activated Treg cells were seen to promote TGF-β1 production, as well as IDO and HO-1 expression by PMNs. These findings suggest that Treg cells might play an important role in the direct control of innate immune responses through the induction of neutrophils with immunosuppressive properties that generate IL-10, TGF-β1, IDO and HO-1., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Heat shock proteins (HSPs) are stress-responsive proteins that serve as important molecules contributing to cellular "protein triage." We and others have reported an increase of selected HSPs in multiple sclerosis (MS) lesions. However, the exact expression pattern of HSP family genes in MS is not known. The aim of our research was to assess global transcriptional changes of all gene members of the HSP families within MS lesions and associated normal-appearing white matter (NAWM). To this end, we used laser capture microdissection (LCM) to isolate defined regions of chronic-active MS lesions (n = 5), one of the most common types of MS lesions. To identify changes in HSP genes in relation to different areas of the plaque, we used genome-wide microarray analysis. We detected a significant change in the transcriptional profile of the demyelinated region compared with NAWM. In particular, overall expression of different HSP genes was upregulated in different areas of chronic-active lesion. These changes were linked to an upregulation of heat shock factor 4 (HSF4). This is the first global analysis of transcriptional changes in HSPs in the central nervous system during MS. The results support a relationship between HSP activation and lesion activity., (Copyright © 2012 Wiley Periodicals, Inc.)

MicroRNAs (miRNAs) are an emerging group of short, noncoding RNAs that play an important role in regulating expression of classical genes. Thus far little is known about their role in autoimmune demyelination. In this study, we analyzed changes in the miRNA profile in CD4(+) T cells that occurred during the recognition of the myelin autoantigen, MOG(35-55). We found that, both in vivo and in vitro, myelin antigen stimulation resulted in significant up-regulation of miR-301a, miR-21, and miR-155. Furthermore, these three miRNAs were overexpressed in T cells infiltrating the CNS in animals with experimental autoimmune encephalomyelitis. Use of specific miRNA antagonists, antagomirs, revealed that miR-301a contributed to the development of the T-helper type 17 subset via targeting the IL-6/23-STAT3 pathway. This contribution appeared to be mediated by the miR-301a effect on the expression of the PIAS3, a potent inhibitor of the STAT3 pathway. Manipulation of miR-301a levels or PIAS3 expression in myelin-specific CD4(+) T cells led to significant changes in the severity of experimental autoimmune encephalomyelitis. Thus, we have identified a role of miR-301a in regulating the function of myelin-reactive T-helper type 17 cells, supporting a role for miR-301a and PIAS3 as candidates for therapeutic targets for controlling of autoimmune demyelination.

Multiple sclerosis is a common disease of the central nervous system in which the interplay between inflammatory and neurodegenerative processes typically results in intermittent neurological disturbance followed by progressive accumulation of disability. Epidemiological studies have shown that genetic factors are primarily responsible for the substantially increased frequency of the disease seen in the relatives of affected individuals, and systematic attempts to identify linkage in multiplex families have confirmed that variation within the major histocompatibility complex (MHC) exerts the greatest individual effect on risk. Modestly powered genome-wide association studies (GWAS) have enabled more than 20 additional risk loci to be identified and have shown that multiple variants exerting modest individual effects have a key role in disease susceptibility. Most of the genetic architecture underlying susceptibility to the disease remains to be defined and is anticipated to require the analysis of sample sizes that are beyond the numbers currently available to individual research groups. In a collaborative GWAS involving 9,772 cases of European descent collected by 23 research groups working in 15 different countries, we have replicated almost all of the previously suggested associations and identified at least a further 29 novel susceptibility loci. Within the MHC we have refined the identity of the HLA-DRB1 risk alleles and confirmed that variation in the HLA-A gene underlies the independent protective effect attributable to the class I region. Immunologically relevant genes are significantly overrepresented among those mapping close to the identified loci and particularly implicate T-helper-cell differentiation in the pathogenesis of multiple sclerosis.

Heat shock protein 70 (Hsp70), a prominent member of the heat shock protein family, is a stress-induced chaperone, contributing to the "protein triage" mechanism. However, we and others have previously shown that chaperonin activity of Hsp70 also promotes immune recognition of protein/peptide antigens, including myelin autoantigens. Hsp70 has been strikingly elevated in multiple sclerosis (MS) lesions. In a search for the mechanism of Hsp70 up-regulation in MS, we analyzed Hsp70 expression in peripheral blood mononuclear cells (PBMCs) from MS patients (n = 49), healthy controls (n = 40), and patients with rheumatoid arthritis, (RA; n = 13). Hsp70 was detected by Western blot, and Hsp70 levels were quantified by ELISA. We found that Hsp70 was expressed at low levels in ex vivo PBMCs. However, after heat shock, Hsp70 was up-regulated significantly more (up to sixfold) in MS patients compared with healthy controls. This significant overproduction of Hsp70 was also seen following another stress condition, LPS stimulation. Hsp70 is a product of several independent genes, and we found the HSPA1B gene product to be the major form responsible for Hsp70 protein overexpression in PBMCs. Hsp70 overexpression was preceded by increased nuclear presence of heat shock factor 1 (HSF1). HSF1 activation depends on phosphorylation, and we found that inhibition of the A group of protein kinase C isoenzymes significantly reduced inducible Hsp70 production. These results indicate that immune cells from MS patients are more prone to Hsp70 induction under stress conditions, suggesting a possible link between Hsp70 overexpression and development of autoimmunity.

Valpha14 invariant (Valpha14i) NKT cells are a subset of regulatory T cells that utilize a semi-invariant TCR to recognize glycolipids associated with monomorphic CD1d molecules. During development in the thymus, CD4(+)CD8(+) Valpha14i NKT precursors recognizing endogenous CD1d-associated glycolipids on other CD4(+)CD8(+) thymocytes are selected to undergo a maturation program involving sequential expression of CD44 and NK-related markers such as NK1.1. The molecular requirements for Valpha14i NKT cell maturation, particularly at early developmental stages, remain poorly understood. In this study, we show that CD4-Cre-mediated T cell-specific inactivation of c-Myc, a broadly expressed transcription factor with a wide range of biological activities, selectively impairs Valpha14i NKT cell development without perturbing the development of conventional T cells. In the absence of c-Myc, Valpha14i NKT cell precursors are blocked at an immature CD44(low)NK1.1(-) stage in a cell autonomous fashion. Residual c-Myc-deficient immature Valpha14i NKT cells appear to proliferate normally, cannot be rescued by transgenic expression of BCL-2, and exhibit characteristic features of immature Valpha14i NKT cells such as high levels of preformed IL-4 mRNA and the transcription factor promyelocytic leukemia zinc finger. Collectively our data identify c-Myc as a critical transcription factor that selectively acts early in Valpha14i NKT cell development to promote progression beyond the CD44(low)NK1.1(-) precursor stage.

Background: Neutrophil cytosolic factor 1, p47phox (NCF1) is a component of the leukocyte NADPH oxidase complex mediating formation of reactive oxygen intermediates (ROI) which play an important role in host defense and autoimmunity. An individual genomic pattern of ncf1 and its two types of pseudogenes (reflected by the DeltaGT/GTGT ratio) may influence the individual capacity to produce ROI., Methods: NCF1DeltaGT/GTGT ratios were correlated with clinical parameters and ROI production during Plasmodium falciparum malaria and with susceptibility to the autoimmune disease multiple sclerosis (MS)., Results: Among Gabonese children with severe malaria, ROI production from peripheral blood tended to be higher in individuals with a DeltaGT/GTGT ratio

Protracted inflammation has been associated with the generation of autoimmune responses. In this respect, increase in the chaperonin, heat shock protein 70 (hsp70) is an outcome of prolonged inflammatory stress. Previous experiments have shown that overexpression of inducible hsp70 in vitro enhanced myelin autoantigen recognition. To prove the role of hsp70 in myelin-directed responses in vivo, we applied a mouse deficient in the major gene encoding inducible hsp70, hsp70.1. Hsp70.1(-/-) mice sensitized for experimental autoimmune encephalomyelitis (EAE) with myelin oligodendrocyte glycoprotein (MOG) peptide 35-55, displayed almost complete resistance to the disease. This correlated with the loss of T cell proliferation and IFN-gamma production in response to MOG(35-55). T cell transfer experiments as well as antigen presentation assays in vitro demonstrated that hsp70 deficiency was associated with dysfunction in the activation of autoreactive T cells. Moreover, T cell responses to ovalbumin (OVA) peptide 323-339 were altered and CD4(+) T cells were more prone to TCR-induced apoptosis, suggesting broader spectrum of T cell defect in hsp70.1(-/-) mice. These results provide compelling evidence for generalized effect mediated by inducible hsp70 in the recognition of myelin self and non-self antigens that influences the cytokine profile of the immune response affecting autoimmune demyelination.

Polymorphisms in the CTLA4 gene region have been associated with susceptibility to autoimmune diseases. The recently described single nucleotide polymorphism CT60, located in the 3' untranslated region of CTLA4 is associated with Graves' disease, thyroiditis, autoimmune diabetes and other autoimmune diseases. A case-control association study was conducted in German, Hungarian and Polish multiple sclerosis (MS) patients and regional control individuals for the CTLA4 CT60 and +49A/G polymorphisms. No significant association of these polymorphisms or respective haplotypes with MS was found. No association of CT60 genotypes with T cell expression of ICOS and CTLA-4 after in vitro stimulation was detected.

Valpha14 invariant natural killer T (Valpha14i NKT) cells are a unique lineage of mouse T cells that share properties with both NK cells and memory T cells. Valpha14i NKT cells recognize CDld-associated glycolipids via a semi-invariant T cell receptor (TCR) composed of an invariant Valpha14-Jalpha 18 chain paired preferentially with a restricted set of TCRbeta chains. During development in the thymus, rare CD4+ CD8+ (DP) cortical thymocytes that successfully rearrange the semi-invariant TCR are directed to the Valpha14i NKT cell lineage via interactions with CD d-associated endogenous glycolipids expressed by other DP thymocytes. As they mature, Valphal4i NKT lineage cells upregulate activation markers such as CD44 and subsequently express NK-related molecules such as NKI.1 and members of the Ly-49 inhibitory receptor family. The developmental program of Valpha l4i NKT cells is critically regulated by a number of signaling cues that have little or no effect on conventional T cell development, such as the Fyn/SAP/SLAM pathway, NFkappaB and T-bet transcription factors, and the cytokine IL-15. The unique developmental requirements of Valphal4i NKT cells may represent a paradigm for other unconventional T cell subsets that are positively selected by agonist ligands expressed on hematopoietic cells.

By combining all the data available from the Genetic Analysis of Multiple sclerosis in EuropeanS (GAMES) project, we have been able to identify 17 microsatellite markers showing consistent evidence for apparent association. As might be expected five of these markers map within the Major Histocompatibility Complex (MHC) and are in LD with HLA-DRB1. Individual genotyping of the 12 non-MHC markers confirmed association for three of them--D11S1986, D19S552 and D20S894. Association mapping across the candidate genes implicated by these markers in 937 UK trio families revealed modestly associated haplotypes in JAG1 (p=0.019) on chromosome 20p12.2 and POU2AF1 (p=0.003) on chromosome 11q23.1.

Invariant Valpha14 (Valpha14i) NKT cells are a murine CD1d-dependent regulatory T cell subset characterized by a Valpha14-Jalpha18 rearrangement and expression of mostly Vbeta8.2 and Vbeta7. Whereas the TCR Vbeta domain influences the binding avidity of the Valpha14i TCR for CD1d-alpha-galactosylceramide complexes, with Vbeta8.2 conferring higher avidity binding than Vbeta7, a possible impact of the TCR Vbeta domain on Valpha14i NKT cell selection by endogenous ligands has not been studied. In this study, we show that thymic selection of Vbeta7(+), but not Vbeta8.2(+), Valpha14i NKT cells is favored in situations where endogenous ligand concentration or TCRalpha-chain avidity are suboptimal. Furthermore, thymic Vbeta7(+) Valpha14i NKT cells were preferentially selected in vitro in response to CD1d-dependent presentation of endogenous ligands or exogenously added self ligand isoglobotrihexosylceramide. Collectively, our data demonstrate that the TCR Vbeta domain influences the selection of Valpha14i NKT cells by endogenous ligands, presumably because Vbeta7 confers higher avidity binding.

Background: There is no consensus method for determining progression of disability in patients with multiple sclerosis (MS) when each patient has had only a single assessment in the course of the disease., Methods: Using data from two large longitudinal databases, the authors tested whether cross-sectional disability assessments are representative of disease severity as a whole. An algorithm, the Multiple Sclerosis Severity Score (MSSS), which relates scores on the Expanded Disability Status Scale (EDSS) to the distribution of disability in patients with comparable disease durations, was devised and then applied to a collection of 9,892 patients from 11 countries to create the Global MSSS. In order to compare different methods of detecting such effects the authors simulated the effects of a genetic factor on disability., Results: Cross-sectional EDSS measurements made after the first year were representative of overall disease severity. The MSSS was more powerful than the other methods the authors tested for detecting different rates of disease progression., Conclusion: The Multiple Sclerosis Severity Score (MSSS) is a powerful method for comparing disease progression using single assessment data. The Global MSSS can be used as a reference table for future disability comparisons. While useful for comparing groups of patients, disease fluctuation precludes its use as a predictor of future disability in an individual.

A humanized mouse bearing the HLA-DR2 (DRA/DRB1*1501) protein associated with multiple sclerosis (MS) and the myelin basic protein (MBP) 85-99-specific HLA-DR2-restricted T cell receptor from an MS patient has been used to examine the effectiveness of modified amino acid copolymers poly(F,Y,A,K)n and poly-(V,W,A,K)n in therapy of MBP 85-99-induced experimental autoimmune encephalomyelitis (EAE) in comparison to Copolymer 1 [Copaxone, poly(Y,E,A,K)n]. The copolymers were designed to optimize binding to HLA-DR2. Vaccination, prevention, and treatment of MBP-induced EAE in the humanized mice with copolymers FYAK and VWAK ameliorated EAE more effectively than Copolymer 1, reduced the number of pathological lesions, and prevented the up-regulation of human HLA-DR on CNS microglia. Moreover, VWAK inhibited MBP 85-99-specific T cell proliferation more efficiently than either FYAK or Copolymer 1 and induced anergy of HLA-DR2-restricted transgenic T cells as its principle mechanism. In contrast, FYAK induced proliferation and a pronounced production of the antiinflammatory T helper 2 cytokines IL-4 and IL-10 from nontransgenic T cells as its principle mechanism of immunosuppression. Thus, copolymers generated by using different amino acids inhibited disease using different mechanisms to regulate T cell responses.

Autoreactive T cells represent a natural repertoire of T cells in both diseased patients and healthy individuals. The mechanisms regulating the function of these autoreactive T cells are still unknown. Ob1A12 is a myelin basic protein (MBP)-reactive Th cell clone derived from a patient with relapsing-remitting multiple sclerosis. Mice transgenic for this human TCR and DRA and DRB1*1501 chains develop spontaneous experimental autoimmune encephalomyelitis. The reactivity of Ob1A12 is reported to be restricted to recognition of MBP peptide 85-99 in the context of DRB1*1501. DRA/DRB1*1501 and the patient's other restriction element, DRA/DRB1*0401, differ significantly in their amino acid sequences. In this study we describe an altered peptide ligand derived from MBP(85-99) with a single amino acid substitution at position 88 (Val to Lys; 88V-->K), that could stimulate the Ob1A12.TCR in the context of both DRA/DRB1*1501 and DRA/DRB1*0401. Analysis of a panel of transfected T cell hybridomas expressing Ob1A12.TCR and CD4 indicated that Ob1A12.TCR cross-reactivity in the context of DRA/DRB1*0401 is critically dependent on the presence of the CD4 coreceptor. Furthermore, we found that activation of Ob1A12.TCR with MBP altered peptide ligand 85-99 88V-->K presented by DRB1*1501 or DRB1*0401 resulted in significant differences in TCR zeta phosphorylation. Our data indicate that injection of altered peptide ligand into patients heterozygous for MHC class II molecules may result in unexpected cross-reactivities, leading to activation of autoreactive T cells.

Using an adoptive transfer model of experimental autoimmune encephalomyelitis (EAE) induced by myelin basic protein (MBP)-reactive lymph node cells (LNC), we have shown that depletion of gammadelta T cells from LNC resulted in diminished severity of EAE in recipient mice, both clinically and histopathologically. The reduced potency of gammadelta T cell-depleted LNC to induce EAE correlated with decreased cell proliferation in response to MBP. The gammadelta T cell effect upon the threshold of MBP-induced LNC proliferation and EAE transfer was restored by reconstitution of gammadelta T cells derived from either MBP-immunized or naive mice, indicating that this effect was not Ag specific. The enhancing effect of gammadelta T cells on MBP-induced proliferation and EAE transfer required direct cell-to-cell contact with LNC. The gammadelta T cell effect upon the LNC response to MBP did not involve a change in expression of the costimulatory molecules CD28, CD40L, and CTLA-4 on TCRalphabeta(+) cells, and CD40, CD80, and CD86 on CD19(+) and CD11b(+) cells. However, depletion of gammadelta T cells resulted in significant reduction in IL-12 production by LNC. That gammadelta T cells enhanced the MBP response and severity of adoptive EAE by stimulating IL-12 production was supported by experiments showing that reconstitution of the gammadelta T cell population restored IL-12 production, and that gammadelta T cell depletion-induced effects were reversed by the addition of IL-12. These results suggest a role for gammadelta T cells in the early effector phase of the immune response in EAE.

Multiple sclerosis, a primary autoimmune disease of the central nervous system has been characterized by the presence of the demyelinating lesions (plaques) in the CNS. To further understand the gene transcription status of the two most common lesions, chronic active and chronic inactive, we have performed a cDNA microarray analysis of these two lesion type. Comparative analysis of differential gene expression of chronic active and inactive lesions have confirmed the existence of a significant difference in the transcriptional profiles of these two lesion types in both marginal and central areas. Different sets of genes were highlighted, including genes of inflammatory characteristics, apoptosis related and stress-induced, indicating their potential role in MS pathogenesis.

In this study, we investigated the role of the inducible form of heat shock protein 70 (hsp70) in the presentation of the major putative autoantigen in multiple sclerosis, myelin basic protein (MBP), in the context of appropriate MHC class II. By coimmunoprecipitation, we found that MBP is associated with hsp70 in APC in an ATP/ADP-dependent manner. Additionally, using confocal microscopy, hsp70 was detected in the endocytic pathway of APC, where it colocalized with MBP and HLA-DR. The immunodominant epitopes of MBP 85-99 and 80-99 were shown to bind selectively and specifically to hsp70 by surface plasmon resonance. The functional significance of MBP interaction with hsp70 was demonstrated by the detection of enhanced responses of an MBP-specific T cell hybridoma to MBP and MBP 80-99 with increasing levels of hsp70 and reduced responses when hsp70 expression was diminished within APC-expressing DRA*0101, DRB1*1501 (DR1501). However, when MBP 85-99 was used as the stimulus, T cell hybridoma responses were not enhanced by hsp70 overexpression within APC, suggesting that hsp70 contributes to Ag processing rather than Ag presentation. The importance of a direct association between MBP and hsp70 in the presentation pathways was demonstrated by enhanced efficacy of MBP presentation by APC transfected with a plasmid vector encoding a fusion hsp70-MBP protein. This is the first report on the involvement of self-inducible hsp70 in MHC class II-dependent autoantigen processing by APC. It implicates that aberrant self hsp expression may lead to the enhancement/modulation of autoimmune responses.

We have performed the first systematic search for MS susceptibility genes completed in the Polish population. This screen was performed using 6000 microsatellite markers typed in pooled DNA from cases (n=200), controls (n=200) and trio families (n=129). Five associated markers are identified, one (D6S2444) from the HLA region and four are from novel regions not previously associated with MS, 2p16 (D2S2153), 3p13 (D3S3568), 7p22 (D7S2521) and 15q26 (D15S649).

cDNA microarray analysis of the regions of pathologically proven different activity of multiple sclerosis lesions was performed. Major differences in gene expression (DGE) occurred between the lesion margin and lesion centre in active lesions studied (57 and 69 genes differentially expressed, respectively), whereas the margins and centres of silent lesions showed markedly reduced heterogeneity (only 11 and two genes differentially expressed, respectively). To compare differences between chronic active and silent lesions, we performed DGE comparison of the pooled data from both types of lesions. The major DGE occurred at the lesion margin, 156 (26; 5%), the greater number representing upregulated genes at the margin of active lesions (15%). Fourteen genes were found to be significantly upregulated in marginal versus central zones in active lesions examined. These genes comprised predominantly inflammation/immune-related factors. We also performed DGE analysis of pooled genes upregulated at the margin of active lesions and found that among the 50 genes showing differences, nine out of 14 were identified in the previous analysis of overlapping differentially expressed genes. Thus this microarray analysis has identified a novel set of genes associated with lesion activity in multiple sclerosis, many of them not previously linked with the disease.

Heat shock proteins (hsp) are known to facilitate the generation of specific immune responses by chaperoning proteins and peptides involved in T cell activation. Hsp have been shown to be strikingly elevated in multiple sclerosis (MS) lesions. The unique chaperonin properties of hsp70 have allowed identification of immunogenic proteins bound to it by the ex vivo demonstration of hsp associations with proteins implicated in the immune response. We have investigated the association of hsp70 with myelin basic protein (MBP), myelin proteolipid protein (PLP) and myelin oligodendrocyte protein (MOG) in MS and control brain tissue. In co-immunoprecipitation experiments, in all samples of MS brains examined (n = 3), but not control brain tissue (n = 3), direct association of MBP with hsp70, but not with hsp90, was found. In some MS brain samples, association between PLP and hsp70 was also seen. In similar co-immunoprecipitation experiments on brain tissue obtained from mice with experimental autoimmune encephalomyelitis (n = 5) induced by immunization with PLP peptide, specific association of hsp70 with PLP and MBP was found. Using surface plasmon resonance we demonstrated specific binding of hsp70 with MBP in vitro. Analysis of the amounts of MBP bound to hsp70 yielded a molecular ratio of MBP binding to hsp70 at 6.5:1. MBP complexed with hsp70 was taken up at significantly higher rates by antigen-presenting cells than MBP alone and enhanced MBP-specific immune responses. These results indicate that hsp70 specifically associates with MBP in MS brain tissue. This association might be relevant to the enhanced immune recognition of MBP in MS.

Intracellular adhesion molecule-1 (ICAM-1) plays an important role in the cascade of adhesion events in the homing of inflammatory cells to the central nervous system (CNS) in experimental autoimmune encephalomyelitis (EAE) and in multiple sclerosis (MS). Two single-base ICAM-1 polymorphisms have been described, in exons 4 and 6, changing codons 241 and 469 in the ICAM-1 gene, respectively. Both polymorphisms result in amino acid changes and can potentially lead to different interactions of ICAM-1 with its ligands. To detect ICAM-1 gene polymorphisms in MS, we have developed a highly sensitive and site-specific, two-stage, nested polymerase chain reaction. Genomic DNA was extracted from blood cells of 79 MS patients and 68 control subjects. The results were confirmed by direct dideoxy chain termination sequencing. The frequency of exon 6 allele T was found to be significantly higher in MS patients than in controls (68% vs 49%). Most interesting, the frequency of exon 6 homozygote K469 was significantly higher in MS patients than in controls (53% vs 34%). Higher frequency of the K469 genotype was found to be independent of possible linkage with the previously described MS susceptibility factor, the HLA class II DR2 allele. In the present study, we have shown for the first time the ICAM-1 gene polymorphisms in MS. The results indicate increased frequency of ICAM-1 exon 6 allele T in MS patients, which may contribute to the MS genetics background.