Publications by authors named "Alexey A Belogurov"

29 Publications

  • Page 1 of 1

At the Cutting Edge against Cancer: A Perspective on Immunoproteasome and Immune Checkpoints Modulation as a Potential Therapeutic Intervention.

Cancers (Basel) 2021 Sep 28;13(19). Epub 2021 Sep 28.

Department of Clinical Sciences and Translational Medicine, University of Rome Tor Vergata, 00133 Rome, Italy.

Immunoproteasome is a noncanonical form of proteasome with enzymological properties optimized for the generation of antigenic peptides presented in complex with class I MHC molecules. This enzymatic property makes the modulation of its activity a promising area of research. Nevertheless, immunotherapy has emerged as a front-line treatment of advanced/metastatic tumors providing outstanding improvement of life expectancy, even though not all patients achieve a long-lasting clinical benefit. To enhance the efficacy of the currently available immunotherapies and enable the development of new strategies, a broader knowledge of the dynamics of antigen repertoire processing by cancer cells is needed. Therefore, a better understanding of the role of immunoproteasome in antigen processing and of the therapeutic implication of its modulation is mandatory. Studies on the potential crosstalk between proteasome modulators and immune checkpoint inhibitors could provide novel perspectives and an unexplored treatment option for a variety of cancers.
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http://dx.doi.org/10.3390/cancers13194852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8507813PMC
September 2021

A New Precision Minimally Invasive Method of Glial Scar Simulation in the Rat Spinal Cord Using Cryoapplication.

Front Surg 2021 15;8:607551. Epub 2021 Jul 15.

Department of Clinical Neurosciences, NCL-Neuromed Institute of Neurosciences, Rome, Italy.

According to the World Health Organization, every year worldwide up to 500,000 people suffer a spinal cord injury (SCI). Various animal biomodels are essential for searching for novel protocols and therapeutic approaches for SCI treatment. We have developed an original model of post-traumatic spinal cord glial scarring in rats through cryoapplication. With this method the low-temperature liquid nitrogen is used for the cryodestruction of the spinal cord tissue. Forty-five Sprague Dawley (SD) non-linear male rats of the Specific-pathogen-free (SPF) category were included in this experimental study. A Th13 unilateral hemilaminectomy was performed with dental burr using an operating microscope. A specifically designed cryogenic probe was applied to the spinal cord for one minute through the created bone defect. The animals were euthanized at different time points ranging from 1 to 60 days after cold-induced injury. Their Th12-L1 vertebrae with the injured spinal cord region were removed "e" for histological examination. Our data demonstrate that cryoapplication producing a topical cooling around-20°C, caused a highly standardized transmural lesion of the spinal cord in the dorsoventral direction. The lesion had an "hour-glass" shape on histological sections. During the entire study period (days 1-60 of the post-trauma period), the necrotic processes and the development of the glial scar (lesion evolution) were contained in the surgically approached vertebral space (Th13). Unlike other known experimental methods of SCI simulation (compression, contusion, etc.), the proposed technique is characterized by minimal invasiveness, high precision, and reproducibility. Also, histological findings, lesion size, and postoperative clinical course varied only slightly between different animals. An original design of the cryoprobe used in the study played a primary role in the achieving of these results. The spinal cord lesion's detailed functional morphology is described at different time points (1-60 days) after the produced cryoinjury. Also, changes in the number of macrophages at distinct time points, neoangiogenesis and the formation of the glial scar's fibrous component, including morphodynamic characteristics of its evolution, are analyzed. The proposed method of cryoapplication for inducing reproducible glial scars could facilitate a better understanding of the self-recovery processes in the damaged spinal cord. It would be evidently helpful for finding innovative approaches to the SCI treatment.
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http://dx.doi.org/10.3389/fsurg.2021.607551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8320592PMC
July 2021

Drift of the Subgingival Periodontal Microbiome during Chronic Periodontitis in Type 2 Diabetes Mellitus Patients.

Pathogens 2021 Apr 22;10(5). Epub 2021 Apr 22.

Ministry of Healthcare of the Russian Federation, A.I. Evdokimov Moscow State University of Medicine and Dentistry, 127473 Moscow, Russia.

Since periodontitis and type 2 diabetes mellitus are complex diseases, a thorough understanding of their pathogenesis requires knowing the relationship of these pathologies with other disorders and environmental factors. In this study, the representability of the subgingival periodontal microbiome of 46 subjects was studied by 16S rRNA gene sequencing and shotgun sequencing of pooled samples. We examined 15 patients with chronic periodontitis (CP), 15 patients with chronic periodontitis associated with type 2 diabetes mellitus (CPT2DM), and 16 healthy subjects (Control). The severity of generalized chronic periodontitis in both periodontitis groups of patients (CP and CPT2DM) was moderate (stage II). The male to female ratios were approximately equal in each group (22 males and 24 females); the average age of the subjects was 53.9 ± 7.3 and 54.3 ± 7.2 years, respectively. The presence of overweight patients (Body Mass Index (BMI) 30-34.9 kg/m) and patients with class 1-2 obesity (BMI 35-45.9 kg/m) was significantly higher in the CPT2DM group than in patients having only chronic periodontitis or in the Control group. However, there was no statistically significant difference in all clinical indices between the CP and CPT2DM groups. An analysis of the metagenomic data revealed that the alpha diversity in the CPT2DM group was increased compared to that in the CP and Control groups. The microbiome biomarkers associated with experimental groups were evaluated. In both groups of patients with periodontitis, the relative abundance of was increased compared to that in the Control group. The CPT2DM group was characterized by a lower relative abundance of / and a higher abundance of compared to those in the CP and Control groups. Furthermore, the CP and CPT2DM groups differed in terms of the relative abundance of (which was decreased in the CPT2DM group compared to CP) and (which was increased in the CPT2DM group compared to CP). In addition, differences in bacterial content were identified by a combination of shotgun sequencing of pooled samples and genome-resolved metagenomics. The results indicate that there are subgingival microbiome-specific features in patients with chronic periodontitis associated with type 2 diabetes mellitus.
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http://dx.doi.org/10.3390/pathogens10050504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8145315PMC
April 2021

Control of Genome through Variative Nature of Histone-Modifying Ubiquitin Ligases.

Biochemistry (Mosc) 2021 Jan;86(Suppl 1):S71-S95

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.

Covalent attachment of ubiquitin residue is not only the proteasomal degradation signal, but also a widespread posttranslational modification of cellular proteins in eukaryotes. One of the most important targets of the regulatory ubiquitination are histones. Localization of ubiquitin residue in different regions of the nucleosome attracts a strictly determined set of cellular factors with varied functionality. Depending on the type of histone and the particular lysine residue undergoing modification, histone ubiquitination can lead both to transcription activation and to gene repression, as well as contribute to DNA repair via different mechanisms. An extremely interesting feature of the family of RING E3 ubiquitin ligases catalyzing histone ubiquitination is the striking structural diversity of the domains providing high specificity of modification very similar initial targets. It is obvious that further elucidation of peculiarities of the ubiquitination system involved in histone modification, as well as understanding of physiological role of this process in the maintenance of homeostasis of both single cells and the entire organism, will substantially expand the possibilities of treating a number of socially significant diseases.
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http://dx.doi.org/10.1134/S0006297921140066DOI Listing
January 2021

In-depth characterization of ubiquitin turnover in mammalian cells by fluorescence tracking.

Cell Chem Biol 2021 Aug 5;28(8):1192-1205.e9. Epub 2021 Mar 5.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Sciences, Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation; Lomonosov Moscow State University, Leninskie Gory, 119991 Moscow, Russian Federation. Electronic address:

Despite almost 40 years having passed from the initial discovery of ubiquitin (Ub), fundamental questions related to its intracellular metabolism are still enigmatic. Here we utilized fluorescent tracking for monitoring ubiquitin turnover in mammalian cells, resulting in obtaining qualitatively new data. In the present study we report (1) short Ub half-life estimated as 4 h; (2) for a median of six Ub molecules per substrate as a dynamic equilibrium between Ub ligases and deubiquitinated enzymes (DUBs); (3) loss on average of one Ub molecule per four acts of engagement of polyubiquitinated substrate by the proteasome; (4) direct correlation between incorporation of Ub into the distinct type of chains and Ub half-life; and (5) critical influence of the single lysine residue K27 on the stability of the whole Ub molecule. Concluding, our data provide a comprehensive understanding of ubiquitin-proteasome system dynamics on the previously unreachable state of the art.
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http://dx.doi.org/10.1016/j.chembiol.2021.02.009DOI Listing
August 2021

Polyamines Counteract Carbonate-Driven Proteasome Stalling in Alkaline Conditions.

Biomolecules 2020 11 24;10(12). Epub 2020 Nov 24.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia.

Cancer cells tend to increase intracellular pH and, at the same time, are known to intensively produce and uptake polyamines such as spermine. Here, we show that various amines, including biogenic polyamines, boost the activity of proteasomes in a dose-dependent manner. Proteasome activity in the classical amine-containing buffers, such as 2-(N-morpholino)ethanesulfonic acid (MES), Tris, (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), glycylglycine, bis-Tris propane, and bicine, has a skewed distribution with a maximum at pH of 7.0-8.0. The activity of proteasomes in buffers containing imidazole and bis-Tris is maintained almost on the same level, in the pH range of 6.5-8.5. The third type of activation is observed in buffers based on the amino acids arginine and ornithine, as well as the natural polyamines spermine and spermidine. Proteasome activity in these buffers is dramatically increased at pH values greater than 7.5. Anionic buffers such as phosphate or carbonate, in contrast, inhibit proteasome activity during alkalization. Importantly, supplementation of a carbonate-phosphate buffer with spermine counteracts carbonate-driven proteasome stalling in alkaline conditions, predicting an additional physiological role of polyamines in maintaining the metabolism and survival of cancer cells.
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http://dx.doi.org/10.3390/biom10121597DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7760842PMC
November 2020

Liquid drop of DNA libraries reveals total genome information.

Proc Natl Acad Sci U S A 2020 11 21;117(44):27300-27306. Epub 2020 Oct 21.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow 117997, Russia;

Conventional "bulk" PCR often yields inefficient and nonuniform amplification of complex templates in DNA libraries, introducing unwanted biases. Amplification of single DNA molecules encapsulated in a myriad of emulsion droplets (emulsion PCR, ePCR) allows the mitigation of this problem. Different ePCR regimes were experimentally analyzed to identify the most robust techniques for enhanced amplification of DNA libraries. A phenomenological mathematical model that forms an essential basis for optimal use of ePCR for library amplification was developed. A detailed description by high-throughput sequencing of amplified DNA-encoded libraries highlights the principal advantages of ePCR over bulk PCR. ePCR outperforms PCR, reduces gross DNA errors, and provides a more uniform distribution of the amplified sequences. The quasi single-molecule amplification achieved via ePCR represents the fundamental requirement in case of complex DNA templates being prone to diversity degeneration and provides a way to preserve the quality of DNA libraries.
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http://dx.doi.org/10.1073/pnas.2017138117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959537PMC
November 2020

Exhaustive Search of the Receptor Ligands by the CyCLOPS (Cytometry Cell-Labeling Operable Phage Screening) Technique.

Int J Mol Sci 2020 Aug 29;21(17). Epub 2020 Aug 29.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS, 117997 Moscow, Russia.

Effective and versatile screening of the peptide ligands capable of selectively binding to diverse receptors is in high demand for the state-of-the-art technologies in life sciences, including probing of specificity of the cell surface receptors and drug development. Complex microenvironment and structure of the surface receptors significantly reduce the possibility to determine their specificity, especially when in vitro conditions are utilized. Previously, we designed a publicly available platform for the ultra-high-throughput screening (uHTS) of the specificity of surface-exposed receptors of the living eukaryotic cells, which was done by consolidating the phage display and flow cytometry techniques. Here, we significantly improved this methodology and designed the fADL-1e-based phage vectors that do not require a helper hyperphage for the virion assembly. The enhanced screening procedure was tested on soluble human leukocyte antigen (HLA) class II molecules and transgenic antigen-specific B cells that express recombinant lymphoid B-cell receptor (BCR). Our data suggest that the improved vector system may be successfully used for the comprehensive search of the receptor ligands in either cell-based or surface-immobilized assays.
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http://dx.doi.org/10.3390/ijms21176258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7504098PMC
August 2020

Multiscale computation delivers organophosphorus reactivity and stereoselectivity to immunoglobulin scavengers.

Proc Natl Acad Sci U S A 2020 09 28;117(37):22841-22848. Epub 2020 Aug 28.

Department of Chemistry, The Scripps Research Institute, La Jolla, CA 92037;

Quantum mechanics/molecular mechanics (QM/MM) maturation of an immunoglobulin (Ig) powered by supercomputation delivers novel functionality to this catalytic template and facilitates artificial evolution of biocatalysts. We here employ density functional theory-based (DFT-b) tight binding and funnel metadynamics to advance our earlier QM/MM maturation of A17 Ig-paraoxonase (WTIgP) as a reactibody for organophosphorus toxins. It enables regulation of biocatalytic activity for tyrosine nucleophilic attack on phosphorus. The single amino acid substitution l-Leu47Lys results in 340-fold enhanced reactivity for paraoxon. The computed ground-state complex shows substrate-induced ionization of the nucleophilic l-Tyr37, now H-bonded to l-Lys47, resulting from repositioning of l-Lys47. Multiple antibody structural homologs, selected by phenylphosphonate covalent capture, show contrasting enantioselectivities for a P-chiral phenylphosphonate toxin. That is defined by crystallographic analysis of phenylphosphonylated reaction products for antibodies A5 and WTIgP. DFT-b analysis using QM regions based on these structures identifies transition states for the favored and disfavored reactions with surprising results. This stereoselection analysis is extended by funnel metadynamics to a range of WTIgP variants whose predicted stereoselectivity is endorsed by experimental analysis. The algorithms used here offer prospects for tailored design of highly evolved, genetically encoded organophosphorus scavengers and for broader functionalities of members of the Ig superfamily, including cell surface-exposed receptors.
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http://dx.doi.org/10.1073/pnas.2010317117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7502716PMC
September 2020

Autoantibodies from SLE patients induce programmed cell death in murine fibroblast cells through interaction with TNFR1 receptor.

Sci Rep 2020 07 7;10(1):11144. Epub 2020 Jul 7.

Laboratory of Molecular Immunogenetics of Cancer, Institute of Gene Biology RAS, Moscow, Russia, 119334.

Various pathological processes are known to be associated with the production of IgG autoantibodies, which have high affinity for self-antigens and often cause tissue injury and the development of autoimmune diseases. However, the mechanism of their cytotoxic activity is not clearly understood yet. Here, we have shown that the action of these autoantibodies on cells expressing TNFR1 (the cell surface receptor for TNFα) can cause both caspase-dependent apoptosis and necroptosis of these cells, with suppression of apoptosis resulting in switching to RIP1-dependent necroptosis. Analysis of necroptotic mechanisms has shown that a critical point of necroptosis is phosphorylation of RIP1 and RIP3 kinases, which is followed by the involvement of lysosomes and mitochondria in this process. The induction of cytotoxicity is initiated by the interaction of autoantibodies with TNFR1, and autoantibodies can therefore be regarded as a new functional ligand for this receptor. The innate immunity protein Tag7 (PGLYRP1) described in our recent studies is also a ligand for TNFR1 and competes with autoantibodies for binding with it. Supposedly, the cytotoxic effect of autoantibodies is one of the factors responsible for autoimmune diseases that lead to tissue injury.
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http://dx.doi.org/10.1038/s41598-020-68088-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340778PMC
July 2020

Probing Surface Membrane Receptors Using Engineered Bacteriophage Bioconjugates.

Bioconjug Chem 2019 05 6;30(5):1500-1506. Epub 2019 May 6.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry RAS , Moscow , Russia , 117997.

Specific recognition of ligands by surface receptors of eukaryotic cells is a fundamental process in sensing of the exogenous environment, including cell-to-cell communication. These interactions are therefore widely probed in both basic studies and drug development to enhance or interrupt them. Here, we designed a high-throughput publicly available platform for visualization and selection of eukaryotic cells according to the specificity of surface-exposed receptors by consolidation of phage display and flow cytometry techniques. Polypeptide ligands for membrane receptors are incorporated into every copy of p3 protein of M13K07 bacteriophage, which is intracellularly biotinylated to further accept PE-Cy7-labled streptavidin. Transgenic antigen-specific B-cells expressing membrane-tethered lymphoid B-cell receptor in a single-chain format interacted with engineered bacteriophages exposing the polypeptide ligand with an unprecedented selectivity of 97% and a false-positive detection value of 2.0%. Multivalent binding of the phage bioconjugates with the receptor provided significantly better specificity and sensitivity allowing application of engineered bacteriophage bioconjugates at a concentration 3 orders of magnitude lower in comparison with synthetic biotinylated peptide. We suggest that the platform described in this work may be applied either for routine staining or characterization of orphan membrane receptors exposed on the surface of living mammalian cells in their native environment.
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http://dx.doi.org/10.1021/acs.bioconjchem.9b00218DOI Listing
May 2019

Autocrine-based selection of ligands for personalized CAR-T therapy of lymphoma.

Sci Adv 2018 11 14;4(11):eaau4580. Epub 2018 Nov 14.

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road MB-10, La Jolla, CA 92037, USA.

We report the development of a novel platform to enhance the efficacy and safety of follicular lymphoma (FL) treatment. Since lymphoma is a clonal malignancy of a diversity system, every tumor has a different antibody on its cell surface. Combinatorial autocrine-based selection is used to rapidly identify specific ligands for these B cell receptors on the surface of FL tumor cells. The selected ligands are used in a chimeric antigen receptor T cell (CAR-T) format for redirection of human cytotoxic T lymphocytes. Essentially, the format is the inverse of the usual CAR-T protocol. Instead of being a guide molecule, the antibody itself is the target. Thus, these studies raise the possibility of personalized treatment of lymphomas using a private antibody binding ligand that can be obtained in a few weeks.
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http://dx.doi.org/10.1126/sciadv.aau4580DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6235538PMC
November 2018

Microfluidic droplet platform for ultrahigh-throughput single-cell screening of biodiversity.

Proc Natl Acad Sci U S A 2017 03 15;114(10):2550-2555. Epub 2017 Feb 15.

Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520

Ultrahigh-throughput screening (uHTS) techniques can identify unique functionality from millions of variants. To mimic the natural selection mechanisms that occur by compartmentalization in vivo, we developed a technique based on single-cell encapsulation in droplets of a monodisperse microfluidic double water-in-oil-in-water emulsion (MDE). Biocompatible MDE enables in-droplet cultivation of different living species. The combination of droplet-generating machinery with FACS followed by next-generation sequencing and liquid chromatography-mass spectrometry analysis of the secretomes of encapsulated organisms yielded detailed genotype/phenotype descriptions. This platform was probed with uHTS for biocatalysts anchored to yeast with enrichment close to the theoretically calculated limit and cell-to-cell interactions. MDE-FACS allowed the identification of human butyrylcholinesterase mutants that undergo self-reactivation after inhibition by the organophosphorus agent paraoxon. The versatility of the platform allowed the identification of bacteria, including slow-growing oral microbiota species that suppress the growth of a common pathogen, , and predicted which genera were associated with inhibitory activity.
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http://dx.doi.org/10.1073/pnas.1621226114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347554PMC
March 2017

Robotic QM/MM-driven maturation of antibody combining sites.

Sci Adv 2016 Oct 19;2(10):e1501695. Epub 2016 Oct 19.

Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road MB-10, La Jolla, CA 92037, USA.

In vitro selection of antibodies from large repertoires of immunoglobulin (Ig) combining sites using combinatorial libraries is a powerful tool, with great potential for generating in vivo scavengers for toxins. However, addition of a maturation function is necessary to enable these selected antibodies to more closely mimic the full mammalian immune response. We approached this goal using quantum mechanics/molecular mechanics (QM/MM) calculations to achieve maturation in silico. We preselected A17, an Ig template, from a naïve library for its ability to disarm a toxic pesticide related to organophosphorus nerve agents. Virtual screening of 167,538 robotically generated mutants identified an optimum single point mutation, which experimentally boosted wild-type Ig scavenger performance by 170-fold. We validated the QM/MM predictions via kinetic analysis and crystal structures of mutant apo-A17 and covalently modified Ig, thereby identifying the displacement of one water molecule by an arginine as delivering this catalysis.
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http://dx.doi.org/10.1126/sciadv.1501695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5072179PMC
October 2016

The Pathogenesis of the Demyelinating Form of Guillain-Barre Syndrome (GBS): Proteo-peptidomic and Immunological Profiling of Physiological Fluids.

Mol Cell Proteomics 2016 07 3;15(7):2366-78. Epub 2016 May 3.

From the ‡Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya str., 16/10, Moscow 117997, Russian Federation; §Moscow Institute of Physics and Technology, Institutskiy pereulok 9, Dolgoprudny 141700, Russian Federation; ‖Research Institute of Physical Chemical Medicine, Malaya Pirogovskaya str., 1a, Moscow 119435, Russian Federation.

Acute inflammatory demyelinating polyneuropathy (AIDP) - the main form of Guillain-Barre syndrome-is a rare and severe disorder of the peripheral nervous system with an unknown etiology. One of the hallmarks of the AIDP pathogenesis is a significantly elevated cerebrospinal fluid (CSF) protein level. In this paper CSF peptidome and proteome in AIDP were analyzed and compared with multiple sclerosis and control patients. A total protein concentration increase was shown to be because of even changes in all proteins rather than some specific response, supporting the hypothesis of protein leakage from blood through the blood-nerve barrier. The elevated CSF protein level in AIDP was complemented by activization of protein degradation and much higher peptidome diversity. Because of the studies of the acute motor axonal form, Guillain-Barre syndrome as a whole is thought to be associated with autoimmune response against neurospecific molecules. Thus, in AIDP, autoantibodies against cell adhesion proteins localized at Ranvier's nodes were suggested as possible targets in AIDP. Indeed, AIDP CSF peptidome analysis revealed cell adhesion proteins degradation, however no reliable dependence on the corresponding autoantibodies levels was found. Proteome analysis revealed overrepresentation of Gene Ontology groups related to responses to bacteria and virus infections, which were earlier suggested as possible AIDP triggers. Immunoglobulin blood serum analysis against most common neuronal viruses did not reveal any specific pathogen; however, AIDP patients were more immunopositive in average and often had polyinfections. Cytokine analysis of both AIDP CSF and blood did not show a systemic adaptive immune response or general inflammation, whereas innate immunity cytokines were up-regulated. To supplement the widely-accepted though still unproven autoimmunity-based AIDP mechanism we propose a hypothesis of the primary peripheral nervous system damaging initiated as an innate immunity-associated local inflammation following neurotropic viruses egress, whereas the autoantibody production might be an optional complementary secondary process.
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http://dx.doi.org/10.1074/mcp.M115.056036DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937510PMC
July 2016

Noggin4 is a long-range inhibitor of Wnt8 signalling that regulates head development in Xenopus laevis.

Sci Rep 2016 Mar 14;6:23049. Epub 2016 Mar 14.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.

Noggin4 is a Noggin family secreted protein whose molecular and physiological functions remain unknown. In this study, we demonstrate that in contrast to other Noggins, Xenopus laevis Noggin4 cannot antagonise BMP signalling; instead, it specifically binds to Wnt8 and inhibits the Wnt/β -catenin pathway. Live imaging demonstrated that Noggin4 diffusivity in embryonic tissues significantly exceeded that of other Noggins. Using the Fluorescence Recovery After Photobleaching (FRAP) assay and mathematical modelling, we directly estimated the affinity of Noggin4 for Wnt8 in living embryos and determined that Noggin4 fine-tune the Wnt8 posterior-to-anterior gradient. Our results suggest a role for Noggin4 as a unique, freely diffusing, long-range inhibitor of canonical Wnt signalling, thus explaining its ability to promote head development.
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http://dx.doi.org/10.1038/srep23049DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4789793PMC
March 2016

Modified siRNA effectively silence inducible immunoproteasome subunits in NSO cells.

Biochimie 2016 Jun 2;125:75-82. Epub 2016 Mar 2.

Institute of Chemical Biology and Fundamental Medicine SB RAS, 8, Lavrentiev Avenue, Novosibirsk, 630090, Russia. Electronic address:

The pathogenesis of autoimmune and neurodegenerative diseases involves overexpression of inducible subunits of the immunoproteasome. However, the clinical application of inhibitors to inducible subunits of the immunoproteasome has been limited due to systemic toxicity. Here, we designed siRNAs that efficiently silence LMP2, LMP7 and MECL-1 gene expression. Inducible subunits of the immunoproteasome are complex siRNA targets because they have a long half-life; therefore, we introduced 2'-O-methyl modifications into nuclease-sensitive sites. This led to 90-95% silencing efficiency and prolonged silencing, eliminating the need for multiple transfections. Furthermore, we showed that in the absence of transfection reagent, siRNAs with lipophilic residues were able to penetrate cells more effectively and decrease the expression of inducible immunoproteasome subunits by 35% after 5 days. These results show that siRNA targeted to inducible immunoproteasome subunits have great potential for the development of novel therapeutics for autoimmune and neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.biochi.2016.02.015DOI Listing
June 2016

Clinical and experimental studies of multiple sclerosis in Russia: experience of the leading national research centers.

Degener Neurol Neuromuscul Dis 2015 6;5:83-90. Epub 2015 Aug 6.

Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow.

Mechanisms of axonal damage and adaptive capacity in multiple sclerosis (MS), including cortical reorganization, have been actively studied in recent years. The lack of regenerative capabilities and the irreversibility of neurodegeneration in MS are critical factors for the optimization of MS treatment. In this study, we present the results of clinical and basic studies in the field of MS by two leading Russian centers. Clinical and neuroimaging correlations show that spinal damage in MS is accompanied by functional reorganization of the cerebral cortex, which is determined not only by the efferent component but also by the afferent component. Comparative analysis of MS treatment with both interferon β1b (IFN-β1b) and IFN-β1a at a dosage of 22 µg for 3 years through subcutaneous administration and glatiramer acetate showed equally high efficiency in reducing the number of exacerbations in relapsing-remitting MS and secondary-progressive MS. We demonstrate a reduced risk of disability in relapsing-remitting MS and secondary-progressive MS patients in all groups treated with IFN-β1 and glatiramer acetate. MS appears to be a disease that would greatly benefit from the development of personalized therapy; thus, adequate molecular predictors of myelin degradation are greatly needed. Therefore, novel ideas related to the viral hypothesis of the etiology of MS and new targets for therapeutic intervention are currently being developed. In this manuscript, we discuss findings of both clinical practice and fundamental research reflecting challenges and future directions of MS treatment in the Russian Federation.
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http://dx.doi.org/10.2147/DNND.S46023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7337142PMC
August 2015

Chemical Polysialylation of Recombinant Human Proteins.

Methods Mol Biol 2015 ;1321:389-404

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry of the RAS, Moscow, Russia.

Design of drug with prolonged therapeutic action is one of the rapid developing fields of modern medical science and required implementation of different methods of protein chemistry and molecular biology. There are several therapeutic proteins needing increasing of their stability, pharmacokinetic, and pharmacodynamics parameters. To make long-live DNA-encoded drug PEGylation was proposed. Alternatively polysialic (colominic) acid, extracted from the cell wall of E. coli, fractionated to the desired size by anion-exchange chromatography and chemically activated to the amine-reactive aldehyde form, may be chemically attached to the polypeptide chain. Conjugates of proteins and polysialic acid generally resemble properties of protein-PEG conjugates, but possess significant negative net charge and are thought to be fully degradable after endocytosis due to the presence of intracellular enzymes, hydrolyzing the polysialic acid. Complete biodegradation of the polysialic acid moiety makes this kind of conjugates preferable for creation of drugs, intended for chronic use. Here, we describe two different protocols of chemical polysialylation. First protocol was employed for the CHO-derived human butyrylcholinesterase with optimized for recovery of specific enzyme activity. Polysialic acid moieties are attached at various lysine residues. Another protocol was developed for high-yield conjugation of human insulin; major conjugation point is the N-terminal residue of the insulin's light chain. These methods may allow to produce polysialylated conjugates of various proteins or polypeptides with reasonable yield and without significant loss of functional activity.
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http://dx.doi.org/10.1007/978-1-4939-2760-9_26DOI Listing
March 2016

Heavy-light chain interrelations of MS-associated immunoglobulins probed by deep sequencing and rational variation.

Mol Immunol 2014 Dec 16;62(2):305-14. Epub 2014 Feb 16.

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia; Faculty of Chemistry, Lomonosov Moscow State University, Moscow, Russia; Institute of Gene Biology, Russian Academy of Sciences, Moscow, Russia. Electronic address:

The mechanisms triggering most of autoimmune diseases are still obscure. Autoreactive B cells play a crucial role in the development of such pathologies and, in particular, production of autoantibodies of different specificities. The combination of deep-sequencing technology with functional studies of antibodies selected from highly representative immunoglobulin combinatorial libraries may provide unique information on specific features in the repertoires of autoreactive B cells. Here, we have analyzed cross-combinations of the variable regions of human immunoglobulins against the myelin basic protein (MBP) previously selected from a multiple sclerosis (MS)-related scFv phage-display library. On the other hand, we have performed deep sequencing of the sublibraries of scFvs against MBP, Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), and myelin oligodendrocyte glycoprotein (MOG). Bioinformatics analysis of sequencing data and surface plasmon resonance (SPR) studies have shown that it is the variable fragments of antibody heavy chains that mainly determine both the affinity of antibodies to the parent autoantigen and their cross-reactivity. It is suggested that LMP1-cross-reactive anti-myelin autoantibodies contain heavy chains encoded by certain germline gene segments, which may be a hallmark of the EBV-specific B cell subpopulation involved in MS triggering.
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http://dx.doi.org/10.1016/j.molimm.2014.01.013DOI Listing
December 2014

Chemical polysialylation of human recombinant butyrylcholinesterase delivers a long-acting bioscavenger for nerve agents in vivo.

Proc Natl Acad Sci U S A 2013 Jan 7;110(4):1243-8. Epub 2013 Jan 7.

Institute of Bioorganic Chemistry Russian Academy of Sciences, 117997 Moscow, Russia.

The creation of effective bioscavengers as a pretreatment for exposure to nerve agents is a challenging medical objective. We report a recombinant method using chemical polysialylation to generate bioscavengers stable in the bloodstream. Development of a CHO-based expression system using genes encoding human butyrylcholinesterase and a proline-rich peptide under elongation factor promoter control resulted in self-assembling, active enzyme multimers. Polysialylation gives bioscavengers with enhanced pharmacokinetics which protect mice against 4.2 LD(50) of S-(2-(diethylamino)ethyl) O-isobutyl methanephosphonothioate without perturbation of long-term behavior.
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http://dx.doi.org/10.1073/pnas.1211118110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3557082PMC
January 2013

Liposome-encapsulated peptides protect against experimental allergic encephalitis.

FASEB J 2013 Jan 9;27(1):222-31. Epub 2012 Oct 9.

M. M. Shemyakin and Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Multiple sclerosis (MS) is a severe inflammatory and neurodegenerative disease with an autoimmune background. Despite the variety of therapeutics available against MS, the development of novel approaches to its treatment is of high importance in modern pharmaceutics. In this study, experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats has been treated with immunodominant peptides of the myelin basic protein (MBP) encapsulated in mannosylated small unilamellar vesicles. The results show that liposome-encapsulated MBP(46-62) is the most effective in reducing maximal disease score during the first attack, while MBP(124-139) and MBP(147-170) can completely prevent the development of the exacerbation stage. Both mannosylation of liposomes and encapsulation of peptides are critical for the therapeutic effect, since neither naked peptides nor nonmannosylated liposomes, loaded or empty, have proved effective. The liposome-mediated synergistic effect of the mixture of 3 MBP peptides significantly suppresses the progression of protracted EAE, with the median cumulative disease score being reduced from 22 to 14 points, compared to the placebo group; prevents the production of circulating autoantibodies; down-regulates the synthesis of Th1 cytokines; and induces the production of brain-derived neurotrophic factor in the central nervous system. Thus, the proposed formulation ameliorates EAE, providing for a less severe first attack and rapid recovery from exacerbation, and offers a promising therapeutic modality in MS treatment.
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http://dx.doi.org/10.1096/fj.12-213975DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3528315PMC
January 2013

Extracellular HspBP1 inhibits formation of a cytotoxic Tag7-Hsp70 complex in vitro and in human serum.

Biochimie 2012 Jan 21;94(1):203-6. Epub 2011 Oct 21.

Institute of Gene Biology, Russian Academy of Sciences (RAS), Moscow 119344, Russia.

Tag7 (PGRP-S) was described as an innate immunity protein. Earlier we have shown that Tag7 forms with Hsp70 a stable complex with cytotoxic and antitumor activity. The same complex is formed in and secreted by cytotoxic T-lymphocytes. We have also found that Hsp-binding protein HspBP1 incapacitates the Tag7-Hsp70 complex. Here we have studied the interaction of extracellular Tag7 and HspBP1. We have shown that HspBP1 binds Tag7 in the conditioned medium of tumor CSML0 cells, thereby preventing formation of the cytotoxic Tag7-Hsp70 complex. We have also found that Tag7, if present in serum (in every third donor on average), is always in complex with HspBP1. This may be a protective measure against indiscriminate attack of the cytotoxic complex on normal cells.
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http://dx.doi.org/10.1016/j.biochi.2011.10.007DOI Listing
January 2012

Reactibodies generated by kinetic selection couple chemical reactivity with favorable protein dynamics.

Proc Natl Acad Sci U S A 2011 Sep 6;108(38):15954-9. Epub 2011 Sep 6.

Institute of Bioorganic Chemistry Russian Academy of Sciences, Moscow 117997, Russia.

Igs offer a versatile template for combinatorial and rational design approaches to the de novo creation of catalytically active proteins. We have used a covalent capture selection strategy to identify biocatalysts from within a human semisynthetic antibody variable fragment library that uses a nucleophilic mechanism. Specific phosphonylation at a single tyrosine within the variable light-chain framework was confirmed in a recombinant IgG construct. High-resolution crystallographic structures of unmodified and phosphonylated Fabs display a 15-Å-deep two-chamber cavity at the interface of variable light (V(L)) and variable heavy (V(H)) fragments having a nucleophilic tyrosine at the base of the site. The depth and structure of the pocket are atypical of antibodies in general but can be compared qualitatively with the catalytic site of cholinesterases. A structurally disordered heavy chain complementary determining region 3 loop, constituting a wall of the cleft, is stabilized after covalent modification by hydrogen bonding to the phosphonate tropinol moiety. These features and presteady state kinetics analysis indicate that an induced fit mechanism operates in this reaction. Mutations of residues located in this stabilized loop do not interfere with direct contacts to the organophosphate ligand but can interrogate second shell interactions, because the H3 loop has a conformation adjusted for binding. Kinetic and thermodynamic parameters along with computational docking support the active site model, including plasticity and simple catalytic components. Although relatively uncomplicated, this catalytic machinery displays both stereo- and chemical selectivity. The organophosphate pesticide paraoxon is hydrolyzed by covalent catalysis with rate-limiting dephosphorylation. This reactibody is, therefore, a kinetically selected protein template that has enzyme-like catalytic attributes.
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http://dx.doi.org/10.1073/pnas.1108460108DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3179052PMC
September 2011

Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross-react with myelin basic protein and EBV antigen.

FASEB J 2011 Dec 22;25(12):4211-21. Epub 2011 Aug 22.

M. M. Shemyakin and Y. A. Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia.

Multiple sclerosis (MS) is a widespread neurodegenerative autoimmune disease with unknown etiology. It is increasingly evident that, together with pathogenic T cells, autoreactive B cells are among the major players in MS development. The analysis of myelin neuroantigen-specific antibody repertoires and their possible cross-reactivity against environmental antigens, including viral proteins, could shed light on the mechanism of MS induction and progression. A phage display library of single-chain variable fragments (scFvs) was constructed from blood lymphocytes of patients with MS as a potential source of representative MS autoantibodies. Structural alignment of 13 clones selected toward myelin basic protein (MBP), one of the major myelin antigens, showed high homology within variable regions with cerebrospinal fluid MS-associated antibodies as well as with antibodies toward Epstein-Barr latent membrane protein 1 (LMP1). Three scFv clones showed pronounced specificity to MBP fragments 65-92 and 130-156, similar to the serum MS antibodies. One of these clones, designated E2, in both scFv and full-size human antibody constructs, was shown to react with both MBP and LMP1 proteins in vitro, suggesting natural cross-reactivity. Thus, antibodies induced against LMP1 during Epstein-Barr virus infection might act as inflammatory trigger by reacting with MBP, suggesting molecular mimicry in the mechanism of MS pathogenesis.
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http://dx.doi.org/10.1096/fj.11-190769DOI Listing
December 2011

Design of targeted B cell killing agents.

PLoS One 2011 6;6(6):e20991. Epub 2011 Jun 6.

M.M. Shemyakin and Yu.A. Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia.

B cells play an important role in the pathogenesis of both systemic and organ-specific autoimmune diseases. Autoreactive B cells not only produce autoantibodies, but also are capable to efficiently present specific autoantigens to T cells. Furthermore, B cells can secrete proinflammatory cytokines and amplify the vicious process of self-destruction. B cell-directed therapy is a potentially important approach for treatment of various autoimmune diseases. The depletion of B cells by anti-CD20/19 monoclonal antibody Retuximab® used in autoimmune diseases therapy leads to systemic side effects and should be significantly improved. In this study we designed a repertoire of genetically engineered B cell killers that specifically affected one kind of cells carrying a respective B cell receptor. We constructed immunotoxins (ITs), fused with c-myc epitope as a model targeting sequence, based on barnase, Pseudomonas toxin, Shiga-like toxin E.coli and Fc domain of human antibody IgGγ1. C-MYC hybridoma cell line producing anti-c-myc IgG was chosen as a model for targeted cell depletion. C-myc sequence fused with toxins provided addressed delivery of the toxic agent to the target cells. We demonstrated functional activity of designed ITs in vitro and showed recognition of the fusion molecules by antibodies produced by targeted hybridoma. To study specificity of the proposed B cells killing molecules, we tested a set of created ITs ex vivo, using C-MYC and irrelevant hybridoma cell lines. Pseudomonas-containing IT showed one of the highest cytotoxic effects on the model cells, however, possessed promiscuous specificity. Shiga-like toxin construct demonstrated mild both cytotoxicity and specificity. Barnase and Fc-containing ITs revealed excellent balance between their legibility and toxic properties. Moreover, barnase and Fc molecules fused with c-myc epitope were able to selectively deplete c-myc-specific B cells and decrease production of anti-c-myc antibodies in culture of native splenocytes, suggesting their highest therapeutic potential as targeted B cell killing agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0020991PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3108985PMC
October 2011

Recognition and degradation of myelin basic protein peptides by serum autoantibodies: novel biomarker for multiple sclerosis.

J Immunol 2008 Jan;180(2):1258-67

Institute of Bioorganic Chemistry, Clinical Hospital, Russian Academy of Sciences, Moscow, Russia.

The pathologic role of autoantibodies in autoimmune disease is widely accepted. Recently, we reported that anti-myelin basic protein (MBP) serum Abs from multiple sclerosis (MS) patients exhibit proteolytic activity toward the autoantigen. The aim of this study is to determine MBP epitopes specific for the autoantibodies in MS and compare these data with those from other neuronal disorders (OND), leading to the generation of new diagnostic and prognostic criteria. We constructed a MBP-derived recombinant "epitope library" covering the entire molecule. We used ELISA and PAGE/surface-enhanced laser desorption/ionization mass spectroscopy assays to define the epitope binding/cleaving activities of autoantibodies isolated from the sera of 26 MS patients, 22 OND patients, and 11 healthy individuals. The levels of autoantibodies to MBP fragments 48-70 and 85-170 as well as to whole MBP and myelin oligodendrocyte glycoprotein molecules were significantly higher in the sera of MS patients than in those of healthy donors. In contrast, selective reactivity to the two MBP fragments 43-68 and 146-170 distinguished the OND and MS patients. Patients with MS (77% of progressive and 85% of relapsing-remitting) but only 9% of patients with OND and no healthy donors were positive for catalysis, showing pronounced epitope specificity to the encephalitogenic MBP peptide 81-103. This peptide retained its substrate properties when flanked with two fluorescent proteins, providing a novel fluorescent resonance energy transfer approach for MS studies. Thus, anti-MBP autoantibody-mediated, epitope-specific binding and cleavage may be regarded as a specific characteristic of MS compared with OND and healthy donors and may serve as an additional biomarker of disease progression.
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http://dx.doi.org/10.4049/jimmunol.180.2.1258DOI Listing
January 2008

Autoantibodies to myelin basic protein catalyze site-specific degradation of their antigen.

Proc Natl Acad Sci U S A 2006 Jan 30;103(2):281-6. Epub 2005 Dec 30.

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10, Miklukho-Maklaya Street, Moscow 117997, Russia.

Autoantibody-mediated tissue destruction is among the main features of organ-specific autoimmunity. This report describes "an antibody enzyme" (abzyme) contribution to the site-specific degradation of a neural antigen. We detected proteolytic activity toward myelin basic protein (MBP) in the fraction of antibodies purified from the sera of humans with multiple sclerosis (MS) and mice with induced experimental allergic encephalomyelitis. Chromatography and zymography data demonstrated that the proteolytic activity of this preparation was exclusively associated with the antibodies. No activity was found in the IgG fraction of healthy donors. The human and murine abzymes efficiently cleaved MBP but not other protein substrates tested. The sites of MBP cleavage determined by mass spectrometry were localized within immunodominant regions of MBP. The abzymes could also cleave recombinant substrates containing encephalytogenic MBP(85-101) peptide. An established MS therapeutic Copaxone appeared to be a specific abzyme inhibitor. Thus, the discovered epitope-specific antibody-mediated degradation of MBP suggests a mechanistic explanation of the slow development of neurodegeneration associated with MS.
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http://dx.doi.org/10.1073/pnas.0509849103DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1324791PMC
January 2006

Catalytic activity of autoantibodies toward myelin basic protein correlates with the scores on the multiple sclerosis expanded disability status scale.

Immunol Lett 2006 Feb 4;103(1):45-50. Epub 2005 Nov 4.

Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, 16/10, Miklukho-Maklaya str., Moscow 117997, Russia.

Autoantibodies toward myelin basic protein (MBP) evidently emerge in sera and cerebrospinal fluid of the patients with multiple sclerosis (MS), as well as in a MS rodent model, i.e., experimental autoimmune encephalomyelitis (EAE). The studies of the last two decades have unveiled somewhat controversial data on the diagnostic applicability of anti-MBP autoantibodies as a disease' marker. Here, we present the results of new functional analysis of the anti-MBP autoantibodies isolated from MS (in patients) and EAE (in mice) sera, based on their proteolytic activity against the targeted autoantigen. The activity was shown to be the intrinsic property of the IgG molecule. No activity was found in the sera-derived antibody fraction of healthy donors and control mice. Sera of 24 patients with clinically proven MS at different stages of the disease, and 20 healthy controls were screened for the anti-MBP antibody-mediated proteolytic activity. The activity correlated with the scores on the MS expanded disability status scale (EDSS) (r(2)=0.85, P<0.001). Thus, the anti-MBP autoantibody-mediated proteolysis may be regarded as an additional marker of the disease progression.
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http://dx.doi.org/10.1016/j.imlet.2005.10.006DOI Listing
February 2006
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