Targeting gut microbiota: new therapeutic opportunities in multiple sclerosis.

Multiple sclerosis (MS) causes long-lasting, multifocal damage to the central nervous system. The complex background of MS is associated with autoimmune inflammation and neurodegeneration processes, and is potentially affected by many contributing factors, including altered composition and function of the gut microbiota. In this review, current experimental and clinical evidence is presented for the characteristics of gut dysbiosis found in MS, as well as for its relevant links with the course of the disease and the dysregulated immune response and metabolic pathways involved in MS pathology. Furthermore, therapeutic implications of these investigations are discussed, with a range of pharmacological, dietary and other interventions targeted at the gut microbiome and thus intended to have beneficial effects on the course of MS.

Ceramide is implicated in humoral peripheral and intrathecal autoimmune response in MS patients.

BACKGROUND: The disturbed metabolism of ceramide (Cer) is supposed to evoke the autoimmune response, contributing to MS pathology. OBJECTIVES: To determine levels of anti-Cer immunoglobulins G (IgGs) in the CSF and serum of subjects with various phenotypes of MS, and to investigate relationships between levels of anti-Cer antibodies and MS-related variables. METHODS: IgGs isolated from serum and the CSF of 68 MS patients and appropriate controls were examined for their reactivity to Cer subspecies. Their levels were compared between the studied groups and compartments, and analyzed with regard to clinical variables. RESULTS: Increased levels of anti-C16:0-, C18:0-, C18:1-, C24:0- and C24:1-Cer IgGs were detected in the CSF and serum of MS patients in comparison with controls. For IgGs against particular Cer subspecies, correlations were found between their CSF and serum level, as well as with the Link index. Serum and the CSF anti-Cer IgGs differed between patients with clinically isolated syndrome (CIS) and relapsing-remitting MS from those with progressive MS. No correlations were found between anti-Cer IgGs and other MS-related clinical variables. CONCLUSION: Patients with MS have shown altered panels of anti-Cer IgGs in the CSF and serum, which might suggest a relevant, though limited role of Cer as a target for autoimmune humoral response. Utility of antibodies against Cer subspecies as potential markers for MS activity and progression deserves further investigations.

Sphingolipid Players in Multiple Sclerosis: Their Influence on the Initiation and Course of the Disease.

Sphingolipids (SLs) play a significant role in the nervous system, as major components of the myelin sheath, contributors to lipid raft formation that organize intracellular processes, as well as active mediators of transport, signaling and the survival of neurons and glial cells. Alterations in SL metabolism and content are observed in the course of central nervous system diseases, including multiple sclerosis (MS). In this review, we summarize the current evidence from studies on SLs (particularly gangliosides), which may shed new light upon processes underlying the MS background. The relevant aspects of these studies include alterations of the SL profile in MS, the role of antibodies against SLs and complexes of SL-ligand-invariant NKT cells in the autoimmune response as the core pathomechanism in MS. The contribution of lipid-raft-associated SLs and SL-laden extracellular vesicles to the disease etiology is also discussed. These findings may have diagnostic implications, with SLs and anti-SL antibodies as potential markers of MS activity and progression. Intriguing prospects of novel therapeutic options in MS are associated with SL potential for myelin repair and neuroprotective effects, which have not been yet addressed by the available treatment strategies. Overall, all these concepts are promising and encourage the further development of SL-based studies in the field of MS.

New Insights into Multiple Sclerosis Mechanisms: Lipids on the Track to Control Inflammation and Neurodegeneration.

Multiple sclerosis (MS) is a central nervous system disease with complex pathogenesis, including two main processes: immune-mediated inflammatory demyelination and progressive degeneration with axonal loss. Despite recent progress in our understanding and management of MS, availability of sensitive and specific biomarkers for these both processes, as well as neuroprotective therapeutic options targeted at progressive phase of disease, are still being sought. Given their abundance in the myelin sheath, lipids are believed to play a central role in underlying immunopathogenesis in MS and seem to be a promising subject of investigation in this field. On the basis of our previous research and a review of the literature, we discuss the current understanding of lipid-related mechanisms involved in active relapse, remission, and progression of MS. These insights highlight potential usefulness of lipid markers in prediction or monitoring the course of MS, particularly in its progressive stage, still insufficiently addressed. Furthermore, they raise hope for new, effective, and stage-specific treatment options, involving lipids as targets or carriers of therapeutic agents.

Distinctive sphingolipid patterns in chronic multiple sclerosis lesions.

Multiple sclerosis (MS) is a CNS disease characterized by immune-mediated demyelination and progressive axonal loss. MS-related CNS damage and its clinical course have two main phases: active and inactive/progressive. Reliable biomarkers are being sought to allow identification of MS pathomechanisms and prediction of its course. The purpose of this study was to identify sphingolipid (SL) species as candidate biomarkers of inflammatory and neurodegenerative processes underlying MS pathology. We performed sphingolipidomic analysis by HPLC-tandem mass spectrometry to determine the lipid profiles in post mortem specimens from the normal-appearing white matter (NAWM) of the normal CNS (nCNS) from subjects with chronic MS (active and inactive lesions) as well as from patients with other neurological diseases. Distinctive SL modification patterns occurred in specimens from MS patients with chronic inactive plaques with respect to NAWM from the nCNS and active MS (Ac-MS) lesions. Chronic inactive MS (In-MS) lesions were characterized by decreased levels of dihydroceramide (dhCer), ceramide (Cer), and SM subspecies, whereas levels of hexosylceramide and Cer 1-phosphate (C1P) subspecies were significantly increased in comparison to NAWM of the nCNS as well as Ac-MS plaques. In contrast, Ac-MS lesions were characterized by a significant increase of major dhCer subspecies in comparison to NAWM of the nCNS. These results suggest the existence of different SL metabolic pathways in the active versus inactive phase within progressive stages of MS. Moreover, they suggest that C1P could be a new biomarker of the In-MS progressive phase, and its detection may help to develop future prognostic and therapeutic strategies for the disease.

Autoimmunity in multiple sclerosis: role of sphingolipids, invariant NKT cells and other immune elements in control of inflammation and neurodegeneration.

Multiple sclerosis (MS) is the most common demyelinating disease of the central nervous system. It is classified as being an autoimmune response in the genetically susceptible individual to a persistent but unidentified antigen(s). Both the adaptive and the innate immune systems are likely to contribute significantly to MS pathogenesis. This review summarizes current understanding of the characteristics of MS autoimmunity in the initiation and progression of the disease. In particular we find it timely to classify the autoimmune responses by focusing on the immunogenic features of myelin-derived lipids in MS including molecular mimicry; on alterations of bioactive sphingolipids mediators in MS; and on functional roles for regulatory effector cells, including innate lymphocyte populations, like the invariant NKT (iNKT) cells which bridge adaptive and innate immune systems. Recent progress in identifying the nature of sphingolipids recognition for iNKT cells in immunity and the functional consequences of the lipid-CD1d interaction opens new avenues of access to the pathogenesis of demyelination in MS as well as design of lipid antigen-specific therapeutics.

A Novel Aza-MBP Altered Peptide Ligand for the Treatment of Experimental Autoimmune Encephalomyelitis.

Myelin basic protein (MBP) is a major target of T cells in lesions of multiple sclerosis (MS) patients and its animal model, experimental autoimmune encephalomyelitis (EAE). Interactions between the major histocompatibility complex II containing antigenic peptides and the T cell receptor activate CD4+ T cells that perpetuate EAE and MS. Previously reported data has shown that treating with an altered peptide ligand (APL) in which the normal antigenic peptide sequence of MBP has been slightly changed at T cell contact positions is helpful in reducing disease in both rodents and humans. The use of natural peptides, which are susceptible to protease degradation, requires high concentrations that can create hypersensitivity reactions. Our hypothesis is that APL containing aza substitutions, CH(R)-N- > N(R)N, could lead to improved protease resistance, reduced clinical disease scores, and a shift in T cell profile. In this study, several aza-APLs and control peptides were synthesized and screened for the best aza-APL candidate (3aza-APL) based on dissociation half time from major histocompatibility complex (MHC) class II, induction of IL-2 response, and resistance to degradation by proteases. The efficacy was then tested in vivo. Results indicate that 3aza-APL is superior to currently available APLs in terms of protease resistance and disease suppression in EAE mice. The 3aza-APL induced anti-inflammatory immune responses by altering key transcription factors and cytokine genes which regulate T cell subpopulations. These data suggest that the novel 3aza-APL has increased protease resistance property and is effective in reducing clinical and physiological signs of disease in EAE animals.

Cytokine-induced release of ceramide-enriched exosomes as a mediator of cell death signaling in an oligodendroglioma cell line.

Th1 pro-inflammatory cytokines, i.e., TNF-α and IFN-γ, in combination are known to induce cell death in several cell types, including oligodendrocytes, but the mechanism of their synergistic cytotoxicity is unclear. Although ceramide (Cer) has been implicated in cytokine- and stress-induced cell death, its intracellular levels alone cannot explain cytokine synergy. We considered the possibility that Cer released as part of extracellular vesicles may contribute to cytokine-induced synergistic cell death. Using a human oligodendroglioma (HOG) cell line as a model, here we show that exosomes derived from TNF-α-treated "donor" cells, while being mildly toxic to fresh cultures (similar to individual cytokines), induce enhanced cell death when added to IFN-γ-primed target cultures in a fashion resembling the effect of cytokine combination. Further, the sphingolipid profiles of secreted exosomes, as determined by HPLC-MS/MS, revealed that the treatment with the cytokines time-dependently induced the formation and exosomal release, in particular of C16-, C24-, and C24:1-Cer species; C16-, C24-, and C24:1-dihydroCer species; and C16-, C24-, and C24:1-SM species. Finally, exogenous C6-Cer or C16-Cer mimicked and enhanced the cytotoxic effects of the cytokines upon HOG cells, thereby supporting the cell death-signaling role of extracellular Cer.

Neuron-microglia interaction induced bi-directional cytotoxicity associated with calpain activation.

Activated microglia release pro-inflammatory factors and calpain into the extracellular milieu, damaging surrounding neurons. However, mechanistic links to progressive neurodegeneration in disease such as multiple sclerosis (MS) remain obscure. We hypothesize that persistent damaged/dying neurons may also release cytotoxic factors and calpain into the media, which then activate microglia again. Thus, inflammation, neuronal damage, and microglia activation, i.e., bi-directional interaction between neurons and microglia, may be involved in the progressive neurodegeneration. We tested this hypothesis using two in vitro models: (i) the effects of soluble factors from damaged primary cortical neurons upon primary rat neurons and microglia and (ii) soluble factors released from CD3/CD28 activated peripheral blood mononuclear cells of MS patients on primary human neurons and microglia. The first model indicated that neurons due to injury with pro-inflammatory agents (IFN-γ) release soluble neurotoxic factors, including COX-2, reactive oxygen species, and calpain, thus activating microglia, which in turn released neurotoxic factors as well. This repeated microglial activation leads to persistent inflammation and neurodegeneration. The released calpain from neurons and microglia was confirmed by the use of calpain inhibitor calpeptin or SNJ-1945 as well as µ- and m-calpain knock down using the small interfering RNA (siRNA) technology. Our second model using activated peripheral blood mononuclear cells, a source of pro-inflammatory Th1/Th17 cytokines and calpain released from auto-reactive T cells, corroborated similar results in human primary cell cultures and confirmed calpain to be involved in progressive MS. These insights into reciprocal paracrine regulation of cell injury and calpain activation in the progressive phase of MS, Parkinson's disease, and other neurodegenerative diseases suggest potentially beneficial preventive and therapeutic strategies, including calpain inhibition.

Invariant natural killer T cells and their ligands: focus on multiple sclerosis.

Invariant natural killer T (iNKT) cells are an innate population of T cells identified by the expression of an invariant T-cell receptor and reactivity to lipid-based antigens complexed with CD1d. They account for a small percentage of lymphocytes, but are extremely potent and play central roles in immunity to infection, in some cancers, and in autoimmunity. The list of relevant stimulatory lipids and glycolipid antigens now includes a range of endogenous self-antigens including the myelin-derived acetylated galactosylceramides. Recent progress in studies to identify the nature of lipid recognition for iNKT cells in autoimmune diseases like multiple sclerosis is likely to foster the development of therapeutic strategies aimed at harnessing iNKT cell activity.

Invariant Natural Killer T-cell anergy to endogenous myelin acetyl-glycolipids in multiple sclerosis.

To extend our studies on glycolipid-reactive invariant Natural Killer T-cell (iNKT-cell) function in multiple sclerosis (MS), we investigated the stimulatory activities of two myelin-derived glycolipids that are poly-acetylated derivatives of ß-galactosylceramide designated as fast-migrating cerebrosides (FMC) by thin-layer chromatography. In healthy subjects, FMC stimulation of peripheral blood cells significantly expanded iNKT-cells similar to α-GalCer and induced significant increases in Th1, Th2 and Th17 cytokines. In marked contrast, MS patients failed to respond to FMCs or to α-GalCer stimulation indicating an anergic response. We propose that myelin-derived FMC glycolipids stimulate iNKT-cell responses in vivo and this is blocked in MS.

Implications of Lymphocyte Anergy to Glycolipids in Multiple Sclerosis (MS): iNKT Cells May Mediate the MS Infectious Trigger.

Immunogenic lipids may play key roles in host defenses against infection and in generating autoimmune inflammation and organ-specific damage. In multiple sclerosis (MS) there are unequivocal autoimmune features and vulnerability to aggravation or induction by microbial or viral infection. We have found glycolipid-driven anergy of circulating lymphocytes in MS indicating that this immune response is affected in MS and the robust effects of iNKT activation with potent cellular and cytokine activities emphasizes its potential importance. Diverse glycolipids including the endogenous myelin acetylated-galactosylceramides (AcGalCer) can drive activation that could be critical to the inflammatory demyelination in the central nervous system and clinical consequences. The iNKT cells and their invariant or iTCR (Vα24Jα18Vß11) receptor an innate defense-a discrete immune arm that is separate from peptide-driven acquired immune responses. This offers new possibilities for insight including a likelihood that the pattern recognition of exogenous microbial and myelin immunogens can overlap and cross-react especially in an inflammatory milieu.

Myelin recovery in multiple sclerosis: the challenge of remyelination.

Multiple sclerosis (MS) is the most common demyelinating and an autoimmune disease of the central nervous system characterized by immune-mediated myelin and axonal damage, and chronic axonal loss attributable to the absence of myelin sheaths. T cell subsets (Th1, Th2, Th17, CD8+, NKT, CD4+CD25+ T regulatory cells) and B cells are involved in this disorder, thus new MS therapies seek damage prevention by resetting multiple components of the immune system. The currently approved therapies are immunoregulatory and reduce the number and rate of lesion formation but are only partially effective. This review summarizes current understanding of the processes at issue: myelination, demyelination and remyelination-with emphasis upon myelin composition/ architecture and oligodendrocyte maturation and differentiation. The translational options target oligodendrocyte protection and myelin repair in animal models and assess their relevance in human. Remyelination may be enhanced by signals that promote myelin formation and repair. The crucial question of why remyelination fails is approached is several ways by examining the role in remyelination of available MS medications and avenues being actively pursued to promote remyelination including: (i) cytokine-based immune-intervention (targeting calpain inhibition), (ii) antigen-based immunomodulation (targeting glycolipid-reactive iNKT cells and sphingoid mediated inflammation) and (iii) recombinant monoclonal antibodies-induced remyelination.

Signaling and regulatory functions of bioactive sphingolipids as therapeutic targets in multiple sclerosis.

Spingolipids (SLs) are an important component of central nervous system (CNS) myelin sheaths and affect the viability of brain cells (oligodendrocytes, neurons and astrocytes) that is determined by signaling mediated by bioactive sphingoids (lyso-SLs). Recent studies indicate that two lipids, ceramide and sphingosine 1-phosphate (S1P), are particularly involved in many human diseases including the autoimmune inflammatory demyelination of multiple sclerosis (MS). In this review we: (1) Discuss possible sources of ceramide in CNS; (2) Summarize the features of the metabolism of S1P and its downstream signaling through G-protein-coupled receptors; (3) Link perturbations in bioactive SLs metabolism to MS neurodegeneration and (4) Compile ceramide and S1P relationships to this process. In addition, we described recent preclinical and clinical trials of therapies targeting S1P signaling, including 2-amino-2-propane-1,3-diol hydrochloride (FTY720, fingolimod) as well as proposed intervention to specify critical SL levels that tilt balances of apoptotic/active ceramide versus anti-apoptotic/inactive dihydroceramide that may offer a novel and important therapeutic approach to MS.

The structural and functional role of myelin fast-migrating cerebrosides: pathological importance in multiple sclerosis.

A family of neutral glycosphingolipids containing a 3-O-acetyl-sphingosine galactosylceramide (3-SAG) has been characterized. Seven new derivatives of galactosylceramide (GalCer), designated as fast-migrating cerebrosides (FMCs) by TLC retention factor, have been identified. The simplest compounds - FMC-1 and FMC-2 - of this series have been characterized as the 3-SAG containing nonhydroxy and hydroxy fatty acyl, respectively. The next two - FMC-3 and FMC-4 - add 6-O-acetyl-galactose and the most complex glycosphingolipids, FMC-5, -6 and -7, are 2,3,4,6-tetra-O-acetyl-3-SAG. These hydrophobic myelin lipid biomarkers coappear with GalCer during myelinogenesis and disappear along with GalCer in de- or dys-myelinating disorders. Myelin lipid antigens, including FMCs, are keys to myelin biology, opening the possibility of new and novel immune modulatory tools for treatment of autoimmune diseases including multiple sclerosis.

ABH blood group antigens in N-glycan of human glycophorin A.

We previously showed that a small proportion of the O-linked oligosaccharide chains of human glycophorin A (GPA) contains blood group A, B or H antigens, relevant to the ABO phenotype of the donor. The structures of these minor O-glycans have been established (Podbielska et al. (2004) [20]). By the use of immunochemical methods we obtained results indicating that ABH blood group epitopes are also present in N-glycan of human GPA (Podbielska and Krotkiewski (2000) [22]). In the present paper we report a detailed analysis of GPA N-glycans using nanoflow electrospray ionization tandem mass spectrometry. N-glycans containing A-, B- and H-related sequences were identified in GPA preparations obtained from erythrocytes of blood group A, B and O donors, respectively. The ABH blood group epitopes are present on one antenna of the N-glycan, whereas a known sialylated sequence NeuAcalpha2-6Galbeta1-4GlcNAc- occurs on the other antenna and other details are in agreement with the known major structure of the GPA N-glycan. In the bulk of the biantennary sialylated N-glycans released from GPA preparations, the blood group ABH epitopes-containing N-glycans, similarly O-glycans, constituted only a minor part. The amount relative to other N-glycans was estimated to 2-6% of blood group H epitope-containing glycans released from GPA-O preparations and 1-2% of blood group A and B epitope-containing glycans, released from GPA-A and GPA-B, respectively.

Novel myelin penta- and hexa-acetyl-galactosyl-ceramides: structural characterization and immunoreactivity in cerebrospinal fluid.

Fast migrating cerebrosides (FMC) are derivatives of galactosylceramide (GalCer). The structures of the most hydrophobic FMC-5, FMC-6, and FMC-7 were determined by electrospray ionization linear ion-trap mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy complementing previous NMR spectroscopy and gas chromatography-mass spectrometry to be 3-O-acetyl-sphingosine-GalCer derivatives with galactose O-acetyl modifications. FMC-5 and FMC-6 are 3-O-acetyl-sphingosine-2,3,4,6-tetra-O-acetyl-GalCer with nonhydroxy and hydroxy-N-fatty-acids, while FMC-7 has an additional O-acetylation of the 2-hydroxy-fatty acid. The immuno-reactivity in human cerebrospinal fluid (CSF) to these acetylated glycolipids was examined in central nervous system (CNS) infectious disease, noninflammatory disorders, and multiple sclerosis (MS). Screening for lipid binding in MS and other neurological disease groups revealed that the greatest anti-hydrophobic FMC reactivity was observed in the inflammatory CNS diseases (meningitis, meningo-encephalitis, and subacute sclerosing panencephalitis). Some MS patients had increased reactivity with the hydrophobic FMCs and with glycoglycerophospholipid MfGL-II from Mycoplasma fermentans. The cross-reactivity of highly acetylated GalCer with microbial acyl-glycolipid raises the possibility that myelin-O-acetyl-cerebrosides, bacterial infection, and neurological disease are linked.

Galactosylation of IgG from rheumatoid arthritis (RA) patients--changes during therapy.

It is well documented that serum IgG from rheumatoid arthritis (RA) patients exhibits decreased galactosylation of its conservative N-glycans (Asn-297) in CH2 domains of the heavy chains; it has been shown that this agalactosylation is proportional to disease severity. In the present investigation we analyzed galactosylation of IgG derived from the patients using a modified ELISA-plate test, biosensor BIAcore and total sugar analysis (GC-MS). For ELISA and BIAcore the binding of IgG preparations, purified from the patients' sera, to two lectins: Ricinus communis (RCA-I) and Griffonia simplicifolia (GSL-II) was applied. Based on ELISA-plate test an agalactosylation factor (AF, a relative ratio of GSL-II/RCA-I binding) was calculated, which was proportional to actual disease severity. Repeated testing of several patients before and after treatment with methotrexate (MTX) alone or in combination with Remicade (a chimeric antibody anti-TNF-alpha) supplied results indicating an increase of IgG galactosylation during the treatment. This introductory observation suggests that IgG galactosylation may be an additional indicator of the RA patients' improvement.

ABH blood group antigens in O-glycans of human glycophorin A.

The major O-linked oligosaccharide structures attached to human glycophorin A (GPA) have been extensively characterized previously. Our own recent findings, obtained by immunochemical methods, suggested the presence of blood group A and B determinants in O-glycans of human glycophorin originating from blood group A or B erythrocytes, respectively. Here, we elucidate the structure of O-glycans, isolated from GPA of blood group A, B, and O individuals by reductive beta-elimination, carrying A, B or H blood group epitopes, respectively. Structural studies based on nanoflow electrospray-ionization tandem mass spectrometry and earlier reported data on the carbohydrate moiety of GPA and ABH antigens allowed us to conclude that these blood group epitopes are elongations of the beta-GlcNAc branch attached to C-6 of the reducing GalNAc. The galactose linked to C-3 of the reducing GalNAc carries NeuAcalpha2-3 linked residue. Identified here O-glycans were found in low amounts, their content estimated at about one percent of all GPA O-glycans. These O-glycans with type-2 core, carrying the blood group A, B or H determinants, have not been identified in GPA so far. Our results demonstrate the efficacy of nanoESI MS/MS in detecting minor oligosaccharide components present in a mixture with much more abundant structures.

[ABO blood-group system: past and present]. / Uklad grupowy ABO: przeszlosc a terazniejszosc.

The ABO blood group system was discovered one hundred years ago; the respective ABH antigens were found in the protein and lipid membrane components of various cells (most importantly, erythrocytes) and soluble blood group substances. The genetics of the ABO system was also elucidated. This old knowledge is still useful, bringing new applications. Although we do not really know the complete biological role of blood group systems, including that of the major ABO system, data, which is still being accumulated, ensure scientists and physicians of its importance in research and the diagnosis and therapy of diseases. For example, susceptibility to some diseases has been shown to be connected with the blood group of the patient, or the phenotype changes of the ABH determinants may indicate the presence of defined form of cancer. The data presented in this article clearly suggest the validity of the ABO blood group system in medical and biological sciences.