Altered serum levels of neuronal proteins and antibodies to them in occupational diseases of the nervous system.

Aclinical and immunological examination of men with occupational pathology, including vibration disease (VD), occupational sensorineural hearing loss (SHL), and chronic mercury intoxication (CMI), was carried out. The comparison group consisted of men comparable in age and total work experience. Serum concentrations of neurotrophins (S100ß, MBP, BDNF) and antibodies (ABs) to S100ß and MBP proteins were determined by enzyme-linked immunosorbent assay. An increase in the level of the S100ß protein was shown in CMI, VD, and a tendency for its increase was found in SHL. In parallel, an increase in AB to the S100ß protein in VD and SHL and a decrease in AB in CMI were noted. A comparative assessment of MBP levels indicated a pronounced increase in its serum concentrations in patients with CMI and VD versus the comparison group. At the same time, an increase in the level of serum ABs to MBP in individuals with VD and SHL, and a decrease in patients with CMI were noted. In patients with CMI, a significant decrease in the BDNF concentration was found, while in SHL and VD, no statistically significant differences were found in comparison with the comparison group. The results obtained confirm importance of assessing serum concentrations of neurotrophic proteins and ABs to them in the case of occupational damage to the nervous system caused by exposure to physical and chemical factors.

Anti-PD1 antibody enhances the anti-tumor efficacy of MUC1-MBP fusion protein vaccine via increasing Th1, Tc1 activity and decreasing the proportion of MDSC in the B16-MUC1 melanoma mouse model.

In previous studies, we have obtained a notable anti-tumor efficacy of the recombinant MUC1-MBP vaccine in the process of mouse B16-MUC1 melanoma treatment. However, the tumor cannot be eliminated completely. We found that the tumor inhibition rate decreased from 81.67% (five immunizations) to 43.67% (eight immunizations) after more than five immunizations, indicating persistent vaccine stimulation may activate immunosuppressive factors. In the present study, we revealed that programmed cell death 1 (PD1), an inhibitory molecule suppressing T cell function, expressed on splenic and tumor-infiltrating T cells were up-regulated by the vaccine. Therefore, to optimize the anti-tumor efficacy of the vaccine, we employed combination immunotherapy with MUC1-MBP vaccine and αPD1 (anti-PD1 antibody). Results showed that combination immunotherapy induced a more remarkable anti-tumor efficacy, the tumor clearance being increased to 80% from 20% which obtain by MUC1-MBP vaccine immunizations. To investigate the possible underlying mechanism, IFN-γ secretion and cytotoxic T lymphocyte (CTL) cytotoxicity were measured by enzyme-linked immunosorbent assay (ELISA) and xCELLigence real-time cell analyzer (RTCA) respectively. T cell subsets and immunosuppressive cells in the mouse spleen and tumor microenvironment were analyzed by FACS. Results showed that the proportion of splenic CD8+T cells and tumor infiltration was increased and the activity of CTL killing, T helper 1 (Th1), Type 1 CD8+T (Tc1) was enhanced, indicating that the anti-tumor efficacy enhanced by combination immunotherapy was mainly through boosting CD8+T cells mediated anti-tumor cellular immunity. Additionally, combination immunotherapy significantly decreased the splenic and tumor-infiltrating myeloid derived suppressor cells (MDSCs). These results demonstrated that combination immunotherapy with MUC1-MBP vaccine and αPD1 was capable to invoke a more potent anti-tumor immune response and provide a foundation for further research.

Elevated mycobacterium avium subsp. paratuberculosis (MAP) antibody titer in Japanese multiple sclerosis.

To investigate whether antibody production against mycobacterium avium subsp. paratuberculosis (MAP) is related to clinical characteristics of multiple sclerosis (MS) and human leukocyte antigen (HLA) alleles, IgG antibody against three MAP peptides and two human peptides homologous to MAP were measured in sera from 103 MS patients and 50 healthy controls (HCs). MS patients had higher IgG levels against MAP2694295-303 (MAP2694-IgG) than HCs, while the other antibodies were comparable. Multivariate analysis demonstrated that higher MAP2694-IgG titers were associated with higher EDSS scores, but not with HLA alleles or dairy product consumption. Immune response against MAP may worsen MS disability.

Multiple Sclerosis: Enzymatic Cross Site-Specific Hydrolysis of H1 Histone by IgGs against H1, H2A, H2B, H3, H4 Histones, and Myelin Basic Protein.

Histones play a key role in chromatin remodeling and gene transcription. Further, free histones in the blood act as damage-associated molecules. Administration of histones to animals results in systemic inflammatory and toxic effects. Myelin basic protein is the principal constituent element of the myelin-proteolipid sheath of axons. Abzymes (antibodies with catalytic activities) are the original features of some autoimmune diseases. In this study, electrophoretically homogeneous IgGs against H1, H2A, H2B, H3, and H4 histones and myelin basic protein (MBP) were isolated from the blood sera of multiple sclerosis (MS) patients by several affinity chromatographies. Using MALDI mass spectrometry, the sites of H1 histone cleavage by IgGs against H1, H2A, H2B, H3, H4, and MBP were determined. It was shown that IgGs against H1 split H1 at 12 sites, while the number of cleavage sites by abzymes against other histones was lower: H2A (9), H2B (7), H3 (3), and H4 (3). The minimum rate of H1 hydrolysis was observed for antibodies against H3 and H4. A high rate of hydrolysis and the maximum number of H1 hydrolysis sites (17) were found for antibodies against MBP. Only a few sites of H1 hydrolysis by anti-H1 antibodies coincided with those for IgGs against H2A, H2B, H3, H4, and MBP. Thus, the polyreactivity of complexation and the enzymatic cross-activity of antibodies against H1, four other histones, and MBP have first been shown. Since histones act as damage molecules, abzymes against histones and MBP can play a negative role in the pathogenesis of MS and probably other different diseases as well.

Assessment of commonly used methods to determine myelin-reactivity of T cells in multiple sclerosis.

Many studies have analyzed myelin-reactivity of T cells in multiple sclerosis (MS); however, with conflicting results. In this study we compare methods to determine myelin reactivity of T cells and aim to delineate the cause of inconsistency in the literature. Challenging T cells with myelin antigens we found a significant increase in antigen-reactivity of T cells from patients with MS using an ELISpot-assay, in contrast to a CFSE-dilution assay. Comparing the two assays showed that the myelin-reactive T cells detected in the ELISpot-assay originated primarily from effector memory T cells in contrast to the myelin-reactive T cells of the CFSE-assay representing a population of both naïve, central memory and effector memory T cells. This diversity in T cell populations activated in the two assays likely contribute to the discrepancy found in the literature and encourages thorough considerations when choosing an assay to determine antigen-specificity of T cells in future studies.

Human umbilical cord mesenchymal stem cell-derived extracellular vesicles attenuate experimental autoimmune encephalomyelitis via regulating pro and anti-inflammatory cytokines.

The therapeutic effects of mesenchymal stem cells-extracellular vesicles have been proved in many inflammatory animal models. In the current study, we aimed to investigate the effect of extracellular vesicles (EVs) derived from human umbilical cord-MSC (hUCSC-EV) on the clinical score and inflammatory/anti-inflammatory cytokines on the EAE mouse model. After induction of EAE in C57Bl/6 mice, they were treated intravenously with hUCSC-EV or vehicle. The clinical score and body weight of all mice was registered every day. On day 30, mice were sacrificed and splenocytes were isolated for cytokine assay by ELISA. Cytokine expression of pro-/anti-inflammatory cytokine by real-time PCR, leukocyte infiltration by hematoxylin and eosin (H&E) staining, and the percent of glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) positive cells by immunohistochemistry were assessed in the spinal cord. Our results showed that hUCSC-EV-treated mice have lower maximum mean clinical score (MMCS), pro-inflammatory cytokines, and inflammatory score in comparison to the control mice. We also showed that hUCSC-EV administration significantly improved body weight and increased the anti-inflammatory cytokines and the frequency of Treg cells in the spleen. There was no significant difference in the percent of GFAP and MBP positive cells in the spinal cord of experimental groups. Finally, we suggest that intravenous administration of hUCSC-EV alleviate induce-EAE by reducing the pro-inflammatory cytokines, such as IL-17a, TNF-α, and IFN-γ, and increasing the anti-inflammatory cytokines, IL-4 and IL-10, and also decrease the leukocyte infiltration in a model of MS. It seems that EVs from hUC-MSCs have the same therapeutic effects similar to EVs from other sources of MSCs, such as adipose or bone marrow MSCs.

Experimental Autoimmune Encephalomyelitis.

Experimental autoimmune encephalomyelitis, originally experimental allergic encephalomyelitis, is the well-known animal model of multiple sclerosis, an immune- mediated, demyelinating, inflammatory chronic disease of the central nervous system. The experimental disease is widely utilized to test new therapies in preclinical studies, to investigate new hypothesis on the possible pathogenic mechanisms of autoimmune reaction directed against the central nervous system or more generally to investigate the interactions between the immune system and the central nervous system that lead to neuroinflammation. The experimental autoimmune encephalomyelitis may be induced following different protocols in mammals, including nonhuman primates, and autoreactive CD4+ T-lymphocytes directed against myelin antigens are the main factors. Here, after introducing the model, we describe the protocol to induce active EAE in inbred mice, we report on a table the different clinical courses of EAE depending on the combination of antigen /mouse strain and we provide indications on how to evaluate the clinics and pathology of this induced disease.

Regulation of EAE by spontaneously generated IL-10-secreting regulatory T cells in HLA-DR15/TCR.Ob1A12 double transgenic mice.

Humanized double transgenic mice express both HLA-DR15 (the MHC gene linked to MS) and TCR.Ob1A12 from a multiple sclerosis patient (that recognizes MBP85-99 presented by HLA-DR15), yet they fail to develop autoimmune encephalomyelitis quickly, although 5-10% develop disease at 12 months. These mice were found to express large numbers of IL-10-secreting splenocytes as early as 4 weeks of age. These regulatory T cells appeared spontaneously without prior immunization with the autoantigen MBP85-99. They were of murine origin and had a cytokine secretion profile and surface phenotype similar to that reported for Tr1 cells. Notably, the frequency of disease appeared to increase at 14 months. The diseased mice had small spleens which averaged 47 mg, while the remaining non-diseased mice in our colony killed at ages 14-15 months had splenocytes that averaged 80 mg (ranging from 47-130 mg). Thus, the appearance of disease was associated with diminution in numbers of IL-10-secreting regulatory T cells with age.

Myelin-specific T cells in animals with Japanese macaque encephalomyelitis.

OBJECTIVE: To determine whether animals with Japanese macaque encephalomyelitis (JME), a spontaneous demyelinating disease similar to multiple sclerosis (MS), harbor myelin-specific T cells in their central nervous system (CNS) and periphery. METHODS: Mononuclear cells (MNCs) from CNS lesions, cervical lymph nodes (LNs) and peripheral blood of Japanese macaques (JMs) with JME, and cervical LN and blood MNCs from healthy controls or animals with non-JME conditions were analyzed for the presence of myelin-specific T cells and changes in interleukin 17 (IL-17) and interferon gamma (IFNγ) expression. RESULTS: Demyelinating JME lesions contained CD4+ T cells and CD8+ T cells specific to myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), and/or proteolipid protein (PLP). CD8+ T-cell responses were absent in JME peripheral blood, and in age- and sex-matched controls. However, CD4+ Th1 and Th17 responses were detected in JME peripheral blood versus controls. Cervical LN MNCs from eight of nine JME animals had CD3+ T cells specific for MOG, MBP, and PLP that were not detected in controls. Mapping myelin epitopes revealed a heterogeneity in responses among JME animals. Comparison of myelin antigen sequences with those of JM rhadinovirus (JMRV), which is found in JME lesions, identified six viral open reading frames (ORFs) with similarities to myelin antigen sequences. Overlapping peptides to these JMRV ORFs did not induce IFNγ responses. INTERPRETATIONS: JME possesses an immune-mediated component that involves both CD4+ and CD8+ T cells specific for myelin antigens. JME may shed new light on inflammatory demyelinating disease pathogenesis linked to gamma-herpesvirus infection.

HIV-Infected Patients: Cross Site-Specific Hydrolysis of H3 and H4 Histones and Myelin Basic Protein with Antibodies against These Three Proteins.

Histones play important roles in chromatin functioning and gene transcription, but in the intercellular space, they are harmful since they stimulate systemic inflammatory and toxic responses. Electrophoretically homogeneous IgGs against myelin basic protein (MBP), as well as H3 and H4 histones, were isolated from sera of HIV-infected patients. In contrast to known classical proteases, these IgGs split exclusively only histones and MBP but no other control proteins. Among 13 sites of hydrolysis of H3 by IgGs against H3 and 14 sites for anti-MBP IgGs, only two sites of the hydrolysis were the same. Between seven cleavage sites of H4 with IgGs against H4 and 9 sites of this histone hydrolysis by antibodies against MBP, only three sites were the same. The sites of hydrolysis of H3 (and H4) with abzymes against these histones and against MBP were different, but several expended protein clusters containing hydrolysis sites are partially overlapped. The existence of enzymatic cross-reactivity of abzymes against H3 and H4 and MBP represents a great menace to humans since due to cell apoptosis, histones constantly occur in human blood. They can hydrolyze MBP of the myelin sheath of axons and play a negative role in the pathogenesis of HIV-infected patients.

HIV-Infected Patients: Cross Site-Specific Hydrolysis of H2a and H2b Histones and Myelin Basic Protein with Antibodies against These Three Proteins.

Anti-DNA antibodies are usually produced against histone-DNA complexes appearing during cell apoptosis, while histones are known as damage-associated molecules. A myelin sheath of axons contains myelin basic protein (MBP) playing an important role in the pathogenesis of autoimmune diseases. Antibodies with enzymatic activities (abzymes) are distinctive features of some autoimmune and viral diseases. Abzymes against different proteins can usually only hydrolyze these specific proteins. Using sequential chromatographies of homogeneous IgG preparations from sera of HIV-infected patients on columns with immobilized MBP, H2a, and H2b histones, the anti-MBP, anti-H2a, and anti-H2b antibodies were obtained. It was first shown that IgGs against H2a and H2b effectively hydrolyze these histones and MBP, while anti-MBP split MBP, H2a, and H2b, but no other control proteins. Using the MALDI mass spectrometry, the cleavage sites of H2a, H2b, and MBP by abzymes against these three proteins were found. Among 14 sites of hydrolysis of H2a by IgGs against H2a and 10 sites by anti-MBP IgGs, only one site of hydrolysis was the same for these abzymes. Eleven cleavage sites of H2b with IgGs against H2b and 10 sites of its hydrolysis with antibodies against MBP were different. Anti-H2a, anti-H2b, and anti-MBP abzymes are unpredictable examples of IgGs possessing not only cross-complexation but also catalytic cross-reactivity, which may be a common phenomenon for such abzymes in patients with different autoimmune diseases. The existence of cross-reactivity of abzymes against H2a and H2b histones and MBP represent a great danger to humans since, in contrast with MBP, histones due to cell apoptosis constantly occur in human blood. Anti-H2a, anti-H2b, and anti-MBP can attack and hydrolyze myelin basic protein of the myelin sheath of axons and plays a negative role in the pathogenesis of several pathologies.

Engineered regulatory T cells expressing myelin-specific chimeric antigen receptors suppress EAE progression.

The expansion of polyclonal T regulatory cells (Tregs) offers great promise for the treatment of immune-mediated diseases, such as multiple sclerosis (MS). However, polyclonal Tregs can be non-specifically immunosuppressive. Based on the advancements with chimeric antigen receptor (CAR) therapy in leukemia, we previously engineered Tregs to express a T-cell receptor (TCR) specific for a myelin basic protein (MBP) peptide. These TCR-engineered specific Tregs suppressed the proliferation of MBP-reactive T effector cells and ameliorated myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). Herein, we extend this approach by creating human regulatory T cells expressing functional single-chain chimeric antigen receptors (scFv CAR), targeting either MBP or MOG. These scFv CAR-transduced Tregs retained FoxP3 and Helios, characteristic of Treg cells, after long-term expansion in vitro. Importantly, these engineered CNS targeting CAR-Tregs were able to suppress autoimmune pathology in EAE, demonstrating that these Tregs have the potential to be used as a cellular therapy for MS patients.

Obinutuzumab-Induced B Cell Depletion Reduces Spinal Cord Pathology in a CD20 Double Transgenic Mouse Model of Multiple Sclerosis.

B cell-depleting therapies have recently proven to be clinically highly successful in the treatment of multiple sclerosis (MS). This study aimed to determine the effects of the novel type II anti-human CD20 (huCD20) monoclonal antibody (mAb) obinutuzumab (OBZ) on spinal cord degeneration in a B cell-dependent mouse model of MS. Double transgenic huCD20xHIGR3 (CD20dbtg) mice, which express human CD20, were immunised with the myelin fusion protein MP4 to induce experimental autoimmune encephalomyelitis (EAE). Both light and electron microscopy were used to assess myelination and axonal pathology in mice treated with OBZ during chronic EAE. Furthermore, the effects of the already established murine anti-CD20 antibody 18B12 were assessed in C57BL/6 wild-type (wt) mice. In both models (18B12/wt and OBZ/CD20dbtg) anti-CD20 treatment significantly diminished the extent of spinal cord pathology. While 18B12 treatment mainly reduced the extent of axonal pathology, a significant decrease in demyelination and increase in remyelination were additionally observed in OBZ-treated mice. Hence, the data suggest that OBZ could have neuroprotective effects on the CNS, setting the drug apart from the currently available type I anti-CD20 antibodies.

Extreme Diversity of IgGs Against Histones, DNA, and Myelin Basic Protein in the Cerebrospinal Fluid and Blood of Patients with Multiple Sclerosis.

It was recently shown that IgGs from sera of multiple sclerosis (MS) patients are active in the hydrolysis of DNA and myelin basic protein (MBP). We first analyzed the relative concentration of antibodies against five histones (H1, H2a, H2b, H3, and H4) in the cerebrospinal fluid (CSF) and serum of patients with MS. The relative concentrations of blood and CSF IgGs against histones and their activity in the hydrolysis of five histones varied greatly from patient to patient. However, all 28 IgG preparations were hydrolyzed from one to five histones. Relative activities and correlation coefficients among the activities of IgGs from serum and CSF in the hydrolysis of five histones (H1, H2a, H2b, H3, and H4), DNA, and MBP were calculated. It was shown that auto-IgGs from CSF and sera of MS patients are extremely heterogeneous in their affinity to histones, MBP, and DNA. The heterogeneity of IgG-abzymes hydrolyzing DNA, MBP, and histones from CSF and sera was also demonstrated using their isoelectrofocusing. The isofocusing profiles DNase, MBP-, and histone-hydrolyzing activities of IgGs may be very different for various individuals, but the total IgG subfractions with all their activities are distributed from pH 3 to 10.

Nanoimmunosensor based on atomic force spectroscopy to detect anti-myelin basic protein related to early-stage multiple sclerosis.

Multiple Sclerosis (MS) is a chronic inflammatory disorder in the central nervous system for which biomarkers for diagnosis still remain unknown. One potential biomarker is the myelin basic protein. Here, a nanoimmunosensor based on atomic force spectroscopy (AFS) successfully detected autoantibodies against the MBP85-99 peptide from myelin basic protein. The nanoimmunosensor consisted of an atomic force microscope tip functionalization with MBP85-99 peptide, which was made to interact with a mica surface coated either with a layer of anti-MBP85-99 (positive control) or samples of cerebrospinal fluid (CSF) from five multiple sclerosis (MS) patients at different stages of the disease and five non-MS subjects. The adhesion forces obtained from AFS pointed to a high concentration of anti-MBP85-99 for the two patients at early stages of relapsing-remitting multiple sclerosis (RRMS), which were indistinguishable from the positive control. In contrast, considerably lower adhesion forces were measured for all the other eight subjects, including three MS patients with longer history of the disease and under treatment, without episodes of acute MS activity. We have also shown that the average adhesion force between MBP85-99 and anti-MBP85-99 is compatible with the value estimated using steered molecular dynamics. Though further tests will be required with a larger cohort of patients, the present results indicate that the nanoimmunosensor may be a simple tool to detect early-stage MS patients and be useful to understand the molecular mechanisms behind MS.

A Coordinated Dance of Effector and Regulatory T Cells that are Reactive to Different Determinants in Autoimmune Response Orchestration.

This article is about my amazing immunological journey for more than a decade with Eli Sercarz as an achaarya, which in Sanskrit means enlightened mentor. My training and routine interactions with colleagues not only at Eli's laboratory but at University of California at Los Angeles, La Jolla Institute for Immunology, Torrey Pines Institute for Molecular Studies, and University of California, San Diego School of Medicine were instrumental in my continued quest to understand how activation of autoimmune inflammatory cells results in pathology and how inflammatory responses are controlled to keep us healthy. I briefly outline different aspects of immune principles related to the immune response to a self-antigen myelin basic protein (MBP) that induces experimental autoimmune encephalomyelitis, a prototype for multiple sclerosis, and how a coordinated interactive network of regulatory T cells (Tregs) CD4+ (CD4 Tregs) and CD8+ (CD8 Tregs) control the anti-MBP response to maintain immune homeostasis. Eli was my mentor, collaborator, and friend, an incredible human being, and dear member of our extended family. This article is written in his memory with unconditional gratitude.

Systemic immunization with altered myelin basic protein peptide produces sustained antidepressant-like effects.

Immune dysregulation, specifically of inflammatory processes, has been linked to behavioral symptoms of depression in both human and rodent studies. Here, we evaluated the antidepressant effects of immunization with altered peptide ligands of myelin basic protein (MBP)-MBP87-99[A91, A96], MBP87-99[A91], and MBP87-99[R91, A96]-in different models of depression and examined the mechanism by which these peptides protect against stress-induced depression. We found that a single dose of subcutaneously administered MBP87-99[A91, A96] produced antidepressant-like effects by decreasing immobility in the forced swim test and by reducing the escape latency and escape failures in the learned helplessness paradigm. Moreover, immunization with MBP87-99[A91, A96] prevented and reversed depressive-like and anxiety-like behaviors that were induced by chronic unpredictable stress (CUS). However, MBP87-99[R91, A96] tended to aggravate CUS-induced anxiety-like behavior. Chronic stress increased the production of peripheral and central proinflammatory cytokines and induced the activation of microglia in the prelimbic cortex (PrL), which was blocked by MBP87-99[A91, A96]. Immunization with MBP-derived altered peptide ligands also rescued chronic stress-induced deficits in p11, phosphorylated cyclic adenosine monophosphate response element binding protein, and brain-derived neurotrophic factor expression. Moreover, microinjections of recombinant proinflammatory cytokines and the knockdown of p11 in the PrL blunted the antidepressant-like behavioral response to MBP87-99[A91, A96]. Altogether, these findings indicate that immunization with altered MBP peptide produces prolonged antidepressant-like effects in rats, and the behavioral response is mediated by inflammatory factors (particularly interleukin-6), and p11 signaling in the PrL. Immune-neural interactions may impact central nervous system function and alter an individual's response to stress.

MS CD49d+CD154+ Lymphocytes Reprogram Oligodendrocytes into Immune Reactive Cells Affecting CNS Regeneration.

The critical aspect in multiple sclerosis (MS) progression involves insufficient regeneration of CNS resulting from deficient myelin synthesis by newly generated oligodendrocytes (OLs). Although many studies have focused on the role of autoreactive lymphocytes in the inflammatory-induced axonal loss, the problem of insufficient remyelination and disease progression is still unsolved. To determine the effect of myelin-specific lymphocytes on OL function in MS patients and in a mouse model of MS, we cultured myelin induced MS CD49d+CD154+ circulating lymphocytes as well as Experimental Autoimmune Encephalomyelitis (EAE) mouse brain-derived T and memory B cells with maturing oligodendrocyte precursor cells (OPCs). We found that myelin-specific CD49d+CD154+ lymphocytes affected OPC maturation toward formation of immune reactive OLs. Newly generated OLs were characterized by imbalanced myelin basic protein (MBP) and proteolipid protein (PLP) production as well as proinflammatory chemokine/cytokine synthesis. The analysis of cellular pathways responsible for OL reprogramming revealed that CD49d+CD154+ lymphocytes affected miRNA synthesis by dysregulation of polymerase II activity. miR-665 and ELL3 turned out to be the main targets of MS myelin-specific lymphocytes. Neutralization of high intracellular miR-665 concentration restored miRNA and MBP/PLP synthesis. Together, these data point to new targets for therapeutic intervention promoting CNS remyelination.

Differential Diagnostics of Active Progressing Multiple Sclerosis Using a Fluorescent Biomarker with Resonance Energy Transfer.

Previous data showed that myelin-reactive autoantibodies found in patients with multiple sclerosis and mice with experimental autoimmune encephalomyelitis recognize and hydrolyze various fragments of myelin basic protein (MBP). Moreover, antibody-mediated cleavage of the encephalithogenic fragment MBP81-103 flanked with two fluorescent proteins can serve as a new biomarker of multiple sclerosis. Here we describe creation of the next generation of this biomarker based on antibody-dependent degradation of a new chemically synthesized fluorescent substrate with resonance energy transfer that contains fluorophore Cy5 and quencher QXL680 separated by MBP81-99 protein (Cy5-MBP81-99-QXL680). This substrate is degraded during incubation with purified antibodies and B cells from patients with multiple sclerosis, but not healthy volunteers.