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.
In a prospective study involving 5340 individuals, humoral and cellular responses revealed magnitude-dependent protection from COVID-19. Antibodies alone significantly decreased infection rates; ...isolated cellular response provided an intermediate level of protection. The lowest COVID-19 incidence was in the double-positive group.
Abstract
Background
During the ongoing coronavirus disease 2019 (COVID-19) pandemic, many individuals were infected with and have cleared the virus, developing virus-specific antibodies and effector/memory T cells. An important unanswered question is what levels of T-cell and antibody responses are sufficient to protect from the infection.
Methods
In 5340 Moscow residents, we evaluated anti–severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) immunoglobulin M (IgM)/immunoglobulin G (IgG) titers and frequencies of the T cells specific to the membrane, nucleocapsid, and spike proteins of SARS-CoV-2, using interferon gamma (IFN-γ) enzyme-linked immunosorbent spot (ELISpot) assay. Additionally, we evaluated the fractions of virus-specific CD4+ and CD8+ T cells using intracellular staining of IFN-γ and interleukin 2 followed by flow cytometry. We analyzed the COVID-19 rates as a function of the assessed antibody and T-cell responses, using the Kaplan–Meier estimator method, for up to 300 days postinclusion.
Results
We showed that T-cell and antibody responses are closely interconnected and are commonly induced concurrently. Magnitudes of both responses inversely correlated with infection probability. Individuals positive for both responses demonstrated the highest levels of protectivity against the SARS-CoV-2 infection. A comparable level of protection was found in individuals with antibody response only, whereas the T-cell response by itself granted only intermediate protection.
Conclusions
We found that the contribution of the virus-specific antibodies to protection against SARS-CoV-2 infection is more pronounced than that of the T cells. The data on the virus-specific IgG titers may be instructive for making decisions in personalized healthcare and public anti–COVID-19 policies.
Clinical Trials Registration. NCT04898140.
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.
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.
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.
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.
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.
Targeting protein therapeutics to specific cells and tissues is a major challenge in modern medicine. Improving the specificity of protein therapeutic delivery will significantly enhance efficiency ...in drug development. One of the promising tools for protein delivery is extracellular vesicles (EVs) that are enveloped by a complex lipid bilayer. EVs are secreted by almost all cell types and possess significant advantages: biocompatibility, stability, and the ability to penetrate the bloodbrain barrier. Overexpression of the vesicular stomatitis virus protein G (VSV-G) was shown to promote EV formation by the producer cell. We have developed an EV-based system for targeted delivery of protein cargoes to antigen-presenting cells (APCs). In this study, we show that attachment of a recombinant llama nanobody -CD206 to the N-terminus of a truncated VSV-G increases the selectivity of EV cargo delivery mainly to APCs. These results highlight the outstanding technological and biomedical potential of EV-based delivery systems for correcting the immune response in patients with autoimmune, viral, and oncological diseases.
Pemphigus vulgaris is a severe, socially significant autoimmune disease associated with autoantibodies to the desmoglein 3 antigen. The disease affects all age groups, beginning at 18 years of age; ...the mortality rate of pemphigus can reach as high as 50%, depending on a patient's age and a number of other factors. There is no highly selective or personalized therapy for pemphigus vulgaris at the moment. One of the well-known therapeutic approaches to the disease is to use rituximab, an anti-CD20 antibody that can help achieve B cell depletion in peripheral blood. To solve the problem of nonspecific elimination of B cells in patients with pemphigus vulgaris, it is reasonable to use specific immunoligands, their choice being based on an assessment of the level of autoantibodies specific to each of the fragments of desmoglein. In this work, the proportion of autoreactive B cells in patients diagnosed with pemphigus vulgaris is found to be 0.09-0.16%; a positive correlation was revealed between the antibody level and the number of autoreactive B cells to various fragments of desmoglein.
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