Background
B lymphocytes play a pivotal regulatory role in the development of the immune response. It was previously shown that deficiency in B regulatory cells (Bregs) or a decrease in their ...anti-inflammatory activity can lead to immunological dysfunctions. However, the exact mechanisms of Bregs development and functioning are only partially resolved. For instance, only a little is known about the structure of their B cell receptor (BCR) repertoires in autoimmune disorders, including multiple sclerosis (MS), a severe neuroinflammatory disease with a yet unknown etiology. Here, we elucidate specific properties of B regulatory cells in MS.
Methods
We performed a prospective study of the transitional Breg (tBreg) subpopulations with the CD19
+
CD24
high
CD38
high
phenotype from MS patients and healthy donors by (i) measuring their content during two diverging courses of relapsing-remitting MS: benign multiple sclerosis (BMS) and highly active multiple sclerosis (HAMS); (ii) analyzing BCR repertoires of circulating B cells by high-throughput sequencing; and (iii) measuring the percentage of CD27
+
cells in tBregs.
Results
The tBregs from HAMS patients carry the heavy chain with a lower amount of hypermutations than tBregs from healthy donors. The percentage of transitional CD24
high
CD38
high
B cells is elevated, whereas the frequency of differentiated CD27
+
cells in this transitional B cell subset was decreased in the MS patients as compared with healthy donors.
Conclusions
Impaired maturation of regulatory B cells is associated with MS progression.
Extracellular vesicles (EVs) represent membrane-enclosed structures that are likely to be secreted by all living cell types in the animal organism, including cells of peripheral (PNS) and central ...nervous systems (CNS). The ability to cross the blood-brain barrier (BBB) provides the possibility not only for various EV-loaded molecules to be delivered to the brain tissues but also for the CNS-to-periphery transmission of these molecules. Since neural EVs transfer proteins and RNAs are both responsible for functional intercellular communication and involved in the pathogenesis of neurodegenerative diseases, they represent attractive diagnostic and therapeutic targets. Here, we discuss EVs' role in maintaining the living organisms' function and describe deviations in EVs' structure and malfunctioning during various neurodegenerative diseases.
Delivering protein therapeutics specifically into target cells and tissues is a promising avenue in medicine. Advancing this process will significantly enhance the efficiency of the designed drugs. ...In this regard, natural membrane-based systems are of particular interest. Extracellular vesicles (EVs), being the bilayer lipid particles secreted by almost all types of cells, have several principal advantages: biocompatibility, carrier stability, and blood-brain barrier penetrability, which make them a perspective tool for protein therapeutic delivery. Here, we evaluate the engineered genetically encoded EVs produced by a human cell line, which allow efficient cargo loading. In the devised system, the protein of interest is captured by self-assembling structures, i.e., "enveloped protein nanocages" (EPN). In their turn, EPNs are encapsulated in fusogenic EVs by the overexpression of vesicular stomatitis virus G protein (VSV-G). The proteomic profiles of different engineered EVs were determined for a comprehensive evaluation of their therapeutic potential. EVs loading mediated by bio-safe Fos-Jun heterodimerization demonstrates an increased efficacy of active cargo loading and delivery into target cells. Our results emphasize the outstanding technological and biomedical potential of the engineered EV systems, including their application in adoptive cell transfer and targeted cell reprogramming.
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.
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.
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.
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.
Abstract Identifying high-affinity antibodies in human serum is challenging due to extremely low number of circulating B cells specific to the desired antigens. Delays caused by a lack of information ...on the immunogenic proteins of viral origin hamper the development of therapeutic antibodies. We propose an efficient approach allowing for enrichment of high-affinity antibodies against pathogen proteins with simultaneous epitope mapping, even in the absence of structural information about the pathogenic immunogens. To screen therapeutic antibodies from blood of recovered donors, only pathogen transcriptome is required to design an antigen polypeptide library, representing pathogen proteins, exposed on the bacteriophage surface. We developed a two-dimensional screening approach enriching lentiviral immunoglobulin libraries from the convalescent or vaccinated donors against bacteriophage library expressing the overlapping set of polypeptides covering the spike protein of SARS-CoV-2. This platform is suitable for pathogen-specific immunoglobulin enrichment and allows high-throughput selection of therapeutic human antibodies.
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.
ABSTRACT
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 patienst 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.—Gabibov, A. G., Belogurov, A. A., Jr. Lomakin, Y. A., Zakharova, M. Y., Avakyan, M. E., Dubrovskaya, V. V., Smirnov, I. V., Ivanov, A. S., Molnar, A. A., Gurtsevitch, V. E., Diduk, S. V., Smirnova, K. V., Avalle, B., Sharanova, S. N., Tramontano, A., Friboulet, A., Boyko, A. N., Ponomarenko, N. A., Tikunova, N. V. Combinatorial antibody library from multiple sclerosis patients reveals antibodies that cross‐react with myelin basic protein and EBV antigen. FASEB J. 25, 4211–4221 (2011). www.fasebj.org
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